Delayed Disease Onset Research Articles

Antidepressants on Multiple Sclerosis: A Review of In Vitro and In Vivo Models.

BackgroundIncreased prevalence of depression has been observed among patients with multiple sclerosis (MS) and correlated with the elevated levels of proinflammatory cytokines and the overall deregulation of monoaminergic neurotransmitters that these patients exhibit. Antidepressants have proved effective not only in treating depression comorbid to MS, but also in alleviating numerous MS symptoms and even minimizing stress-related relapses. Therefore, these agents could prospectively prove beneficial as a complementary MS therapy.ObjectiveThis review aims at illustrating the underlying mechanisms involved in the beneficial clinical effects of antidepressants observed in MS patients.MethodsThrough a literature search we screened and comparatively assessed papers on the effects of antidepressant use both in vitro and in vivo MS models, taking into account a number of inclusion and exclusion criteria.Results In vitro studies indicated that antidepressants promote neural and glial cell viability and differentiation, reduce proinflammatory cytokines and exert neuroprotective activity by eliminating axonal loss. In vivo studies confirmed that antidepressants delayed disease onset and alleviated symptoms in Experimental Autoimmune Encephalomyelitis (EAE), the most prevalent animal model of MS. Further, antidepressant agents suppressed inflammation and restrained demyelination by decreasing immune cell infiltration of the CNS.ConclusionAntidepressants were efficient in tackling numerous aspects of disease pathophysiology both in vitro and in vivo models. Given that several antidepressants have already proved effective in clinical trials on MS patients, the inclusion of such agents in the therapeutic arsenal of MS should be seriously considered, following an individualized approach to minimize the adverse events of antidepressants in MS patients.

CD11c+CD88+CD317+ myeloid cells are critical mediators of persistent CNS autoimmunity

Natalizumab, a humanized monoclonal antibody (mAb) against α4-integrin, reduces the number of dendritic cells (DC) in cerebral perivascular spaces in multiple sclerosis (MS). Selective deletion of α4-integrin in CD11c+ cells should curtail their migration to the central nervous system (CNS) and ameliorate experimental autoimmune encephalomyelitis (EAE). We generated CD11c.Cre+/-ITGA4fl/fl C57BL/6 mice to selectively delete α4-integrin in CD11c+ cells. Active immunization and adoptive transfer EAE models were employed and compared with WT controls. Multiparameter flow cytometry was utilized to immunophenotype leukocyte subsets. Single-cell RNA sequencing was used to profile individual cells. α4-Integrin expression by CD11c+ cells was significantly reduced in primary and secondary lymphoid organs in CD11c.Cre+/-ITGA4fl/fl mice. In active EAE, a delayed disease onset was observed in CD11c.Cre+/-ITGA4fl/fl mice, during which CD11c+CD88+ cells were sequestered in the blood. Upon clinical EAE onset, CD11c+CD88+ cells appeared in the CNS and expressed CD317+ In adoptive transfer experiments, CD11c.Cre+/-ITGA4fl/fl mice had ameliorated clinical disease phenotype associated with significantly diminished numbers of CNS CD11c+CD88+CD317+ cells. In human cerebrospinal fluid from subjects with neuroinflammation, microglia-like cells display coincident expression of ITGAX (CD11c), C5AR1 (CD88), and BST2 (CD317). In mice, we show that only activated, but not naïve microglia expressed CD11c, CD88, and CD317. Finally, anti-CD317 treatment prior to clinical EAE substantially enhanced recovery in mice.

Adipose-derived stem cells protect motor neurons and reduce glial activation in both in vitro and in vivo models of ALS

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative condition for which new therapeutic options are urgently needed. We injected GFP+ adipose-derived stem cells (EGFP-ADSCs) directly into the cerebrospinal fluid (CSF) of transgenic SOD1G93A mice, a well-characterized model of familial ALS. Despite short-term survival of the injected cells and limited engraftment efficiency, EGFP-ADSCs improved motor function and delayed disease onset by promoting motor neuron (MN) survival and reducing glial activation. We then tested the in vitro neuroprotective potential of mouse ADSCs in astrocyte/MN co-cultures where ALS astrocytes show neurotoxicity. ADSCs were able to rescue MN death caused by ALS astrocytes derived from symptomatic SOD1G93A mice. Further, ADSCs were found to reduce the inflammatory signature of ALS astrocytes by inhibiting the release of pro-inflammatory mediators and inducing the secretion of neuroprotective factors. Finally, mouse ADSCs were able to protect MNs from the neurotoxicity mediated by human induced astrocytes (iAstrocytes) derived from patients with either sporadic or familial ALS, thus for the first time showing the potential therapeutic translation of ADSCs across the spectrum of human ALS. These data in two translational models of ALS show that, through paracrine mechanisms, ADSCs support MN survival and modulate the toxic microenvironment that contributes to neurodegeneration in ALS.

Chloroquine, an Anti-Malaria Drug as Effective Prevention for Hantavirus Infections

We investigated whether chloroquine can prevent hantavirus infection and disease in vitro and in vivo, using the Hantaan virus newborn C57BL/6 mice model and the Syrian hamster model for Andes virus. In vitro antiviral experiments were performed using Vero E6 cells, and Old World and New World hantavirus species. Hantavirus RNA was detected using quantitative RT-PCR. For all hantavirus species tested, results indicate that the IC50 of chloroquine (mean 10.2 ± 1.43 μM) is significantly lower than the CC50 (mean 260 ± 2.52 μM) yielding an overall selectivity index of 25.5. We also investigated the potential of chloroquine to prevent death in newborn mice after Hantaan virus infection and its antiviral effect in the hantavirus Syrian hamster model. For this purpose, C57Bl/6 mother mice were treated subcutaneously with daily doses of chloroquine. Subsequently, 1-day-old suckling mice were inoculated intracerebrally with 5 x 102 Hantaan virus particles. In litters of untreated mothers, none of the pups survived challenge. The highest survival rate (72.7% of pups) was found when mother mice were administered a concentration of 10 mg/kg chloroquine. Survival rates declined in a dose-dependent manner, with 47.6% survival when treated with 5 mg/kg chloroquine, and 4.2% when treated with 1 mg/kg chloroquine. Assessing the antiviral therapeutic and prophylactic effect of chloroquine in the Syrian hamster model was done using two different administration routes (intraperitoneally and subcutaneously using an osmotic pump system). Evaluating the prophylactic effect, a delay in onset of disease was noted and for the osmotic pump, 60% survival was observed. Our results show that chloroquine can be highly effective against Hantaan virus infection in newborn mice and against Andes virus in Syrian hamsters.

ACT-1004-1239, a first-in-class CXCR7 antagonist with both immunomodulatory and promyelinating effects for the treatment of inflammatory demyelinating diseases.

Current strategies for the treatment of demyelinating diseases such as multiple sclerosis (MS) are based on anti-inflammatory or immunomodulatory drugs. Those drugs have the potential to reduce the frequency of new lesions but do not directly promote remyelination in the damaged central nervous system (CNS). Targeting CXCR7 (ACKR3) has been postulated as a potential therapeutic approach in demyelinating diseases, leading to both immunomodulation by reducing leukocyte infiltrates and promyelination by enhancing myelin repair. ACT-1004-1239 is a potent, selective, insurmountable, and orally available first-in-class CXCR7 receptor antagonist. The effect of ACT-1004-1239 was evaluated in the myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) and the cuprizone-induced demyelination mouse models. In addition, ACT-1004-1239 was assessed in a rat oligodendrocyte precursor cell (OPC) differentiation assay in vitro. In the MOG-induced EAE model, ACT-1004-1239 treatment (10-100mg/kg, twice daily, orally) showed a significant dose-dependent reduction in disease clinical scores, resulting in increased survival. At the highest dose tested (100mg/kg, twice daily), ACT-1004-1239 delayed disease onset and significantly reduced immune cell infiltrates into the CNS and plasma neurofilament light chain concentration. Treatment with ACT-1004-1239 dose-dependently increased plasma CXCL12 concentration, which correlated with a reduction of the cumulative disease score. Furthermore, in the cuprizone model, ACT-1004-1239 treatment significantly increased the number of mature myelinating oligodendrocytes and enhanced myelination in vivo. In vitro, ACT-1004-1239 promoted the maturation of OPCs into myelinating oligodendrocytes. These results provide evidence that ACT-1004-1239 both reduces neuroinflammation and enhances myelin repair substantiating the rationale to explore its therapeutic potential in a clinical setting.

Chronic Intermittent Mild Whole-Body Hypothermia Is Therapeutic in a Mouse Model of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that causes motor neuron degeneration. There are no cures or effective treatments for ALS. Therapeutic hypothermia is effectively used clinically to mitigate mortality in patients with acute acquired brain injury and in surgical settings to minimize secondary brain injury. The efficacy of therapeutic hypothermia in chronic neurodegenerative disorders has not been examined. We tested the hypothesis that mild hypothermia/cold acclimation is therapeutic in a transgenic mouse model of ALS caused by expression of mutated human superoxide dismutase-1 gene. At presymptomatic stages of disease, body temperatures (oral and axial) of mutant male mice were persistently hyperthermic (38–38.5 °C) compared to littermate controls, but at end-stage disease mice were generally hypothermic (36–36.5 °C). Presymptomatic mutant mice (awake-freely moving) were acclimated to systemic mild hypothermia using an environmentally controlled chamber (12 h-on/12-off or 24 h-on/24 h-off) to lower body temperature (1–3 °C). Cooled ALS mice showed a significant delay in disease onset (103–112 days) compared to normothermia mice (80–90 days) and exhibited significant attenuation of functional decline in motor performance. Cooled mice examined at 80 days had reduced motor neuron loss, mitochondrial swelling, and spinal cord inflammation compared to non-cooled mice. Cooling attenuated the loss of heat-shock protein 70, mitochondrial uncoupling protein-3, and sumoylated-1 (SUMO1)-conjugated proteins in skeletal muscle and disengaged the mitochondrial permeability transition pore. Cooled ALS mice had a significant extension of lifespan (148 ± 7 days) compared to normothermic mice (135 ± 4 days). Thus, intermittent systemic mild hypothermia is therapeutic in mouse ALS with protective effects manifested within the CNS and skeletal muscle that target mitochondria.

A bioavailable form of curcumin, in combination with vitamin-D- and omega-3-enriched diet, modifies disease onset and outcomes in a murine model of collagen-induced arthritis

ObjectiveCurcumin (CUR), vitamin D3 (D3), and omega-3-fatty acids (O3FA) individually modulate inflammation and pain in arthritis. Although these supplements are widely used, their combinatorial effects have not been defined. In this study, we examined the effects of a D3 and O3FA (VO)-enriched diet in conjunction with a highly bioavailable form of CUR (Cureit/Acumin™) in a collagen-induced arthritis (CIA) murine model.MethodsMale DBA/1J mice were acclimatized to VO-enriched diet and challenged with bovine collagen II (CII). Bioavailable CUR was administered daily by oral gavage from the onset of CII challenge. Disease severity was determined by monitoring joint thickness and standardized clinical score. Cellular infiltration and cartilage degradation in the joints were assessed by histology, serum cytokines profiled by Meso Scale Discovery multiplex assay, and joint matrix metalloproteinases examined by western blots.ResultsCUR by itself significantly decreased disease severity by ~ 60%. Administration of CUR in CIA mice taking a VO-enriched diet decreased disease severity by > 80% and maximally delayed disease onset and progression. Some of the disease-modifying effects was mediated by CUR alone, e.g., suppression of serum anti-collagen antibodies and decrease of cellular infiltration and MMP abundance in the joints of CIA mice. Although CUR alone suppressed inflammatory cytokines in serum of CIA mice, the combination of CUR and VO diet significantly enhanced the suppression (> 2-fold compared to CUR) of TNF, IFN-γ, and MCP-1, all known to be associated with RA pathogenesis.ConclusionThis study provides proof-of-concept that the combination of bioavailable CUR, vitamin D3, and O3FA substantially delays the development and severity of CIA. These findings provide a rationale for systematically evaluating these widely available supplements in individuals at risk for developing future RA.

In Search of an Association Between Genotype and Phenotype in Hereditary Angioedema due to C1-INH Deficiency.

Hereditary angioedema due to C1 inhibitor deficiency (C1-INH-HAE) is caused by mutations affecting the SERPING1 gene. Adult patients (≥ 18 years old) diagnosed with C1-INH-HAE were clustered according to a modified SERPING1 gene mutation classification [5]. Demographic, clinical, and laboratory data were studied. Published manuscripts on the genotype/phenotype relationship were reviewed. Eighty-eight patients participated in the study, with 78 having a classifiable mutation. We compared the data in the 3 largest groups: class 0 only (n = 32), class II only (n = 18), class III only (n = 22). Antigenic C4 and C1 inhibitors were higher in class II (p = 0.008 and p = 0.02, respectively), and facial attacks in the last 6months were more frequent in class III (p = 0.04)). All the other differences were non-significant. Twelve manuscripts on phenotype/genotype correlation were found: missense mutations in SERPING1 gene were associated with delay in disease onset and lower severity score in some studies, whereas the CC F12-C46T/C polymorphism produced earlier disease onset. Our study shows minimal differences regarding clinical phenotype in patients with class 0, II, and III SERPING1 gene mutations, with a tendency to class III having a more severe phenotype. The study should be performed in a larger sample to confirm if they are significant.We propose that larger multicenter, international studies are performed, comparing different SERPING1 gene mutation classifications, combining polymorphisms in other involved genes (kallikrein-kinin system, regulation of vasculature, plasminogen activation) and using different variables and clinical scores to assess C1-INH-HAE disease activity and/or severity in order to study possible genotype/phenotype relationships.

Myeloid TBK1 Deficiency Induces Motor Deficits and Axon Degeneration Through Inflammatory Cell Infiltration.

TANK-binding kinase1 (TBK1) haploinsufficiency has been shown to cause both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD); however, the mechanism is unclear. Myeloid Tbk1 knockout mice (Tbk1-LKO mice) were established and motor function and pathological analyses were also performed. The level of p-TBK1 was analyzed in the ALS animal model and in patient samples using flow cytometry. The expression of inflammatory proteins and mRNAs was analyzed via western blotting and RT-PCR, respectively. The latency to fall in seven-month-old Tbk1-LKO mice was significantly reduced in evaluations conducted on two consecutive days. Overall, 25.6% of Tbk1-LKO mice presented paralysis symptoms and signs, along with a loosened myelin sheath and axon degeneration at 14-16 months of age. Furthermore, the Tbk1 deficiency in myeloid cells induced inflammatory cell infiltration and dysbacteriosis in the digestive tract. Additionally, p-TBK1 levels were reduced by 29.5% and 14.8% in monocytes of patients with definite ALS and probable ALS and decreased by 27.6% and 45.5% in monocytes and microglia of ALS animals, respectively. The use of PEI-mannose-TBK1 or PEI-mannose-SaCas9-sgRNA to delete mutant SOD1 in macrophages significantly delayed disease onset and prolonged survival in the mouse model of ALS. Based on these data, inflammatory monocyte and macrophage infiltration and impaired innate immune defenses contribute to ALS and FTD.

Bilirubin nanomedicine ameliorates the progression of experimental autoimmune encephalomyelitis by modulating dendritic cells

Although the cause of multiple sclerosis (MS) is unclear, an autoimmune attack on myelin-based coating layers of nerve cells in the brain and spinal cord is the main feature of the disease, highlighting modulation of the immune response to myelin as a feasible therapeutic approach. Here, we report the potential of bilirubin nanoparticles (BRNPs) based on the endogenous antioxidant and anti-inflammatory agent, bilirubin, as a therapeutic nanomedicine for MS. In a mouse model of experimental autoimmune encephalomyelitis (EAE), multiple intravenous injections of BRNPs significantly delayed disease onset and suppressed disease progression and severity as well as disease incidence rate without systemic immunosuppression. Following intravenous injection, BRNPs accumulated more extensively and were retained longer in secondary lymphoid organs of EAE-induced mice compared with non-immunized control mice, including in inguinal lymph nodes (iLNs) and spleens, where antigen presenting cells (APCs) activated by the myelin antigen are abundant. Studies of the underlying mechanism of action further revealed that BRNPs negatively regulated the differentiation of naïve CD4+ T cells into T helper 17 (Th17) cells by inhibiting maturation of APCs through scavenging of reactive oxygen species (ROS) overproduced in both dendritic cells (DCs) and macrophages upon antigen uptake. These findings indicate that BRNPs have the potential to be used as a new therapeutic nanomedicine for treatment of various CD4+ T cell-associated autoimmune diseases.

Supranutritional selenium suppresses ROS-induced generation of RANKL-expressing osteoclastogenic CD4+ Tcells and ameliorates rheumatoid arthritis.

ObjectiveThe benefit of Se supplementation in rheumatoid arthritis (RA) has been tested in clinical trials, but results remain inconclusive. The objective of this study was to specifically investigate the potential benefit of supranutritional Se by examining human samples from an area with supranutritional Se intake and testing a mouse model of RA.MethodsPeripheral blood mononuclear cells (PBMCs) from RA patients (N = 57) and healthy controls (HC, N = 71) from an area of supranutritional Se intake (Enshi, Hubei, China) were analysed by flow cytometry. Serum cytokine and Se levels were measured by cytometric beads array (CBA) and inductively coupled plasma mass spectrometry (ICP‐MS), respectively. With sufficient or supranutritional selenium intake, mice were induced with collagen‐induced arthritis (CIA) and examined for disease activity and immunopathology. The influence of Se supplementation in the generation of RANKL‐expressing osteoclastogenic CD4+ T cells was investigated by in vitro assays.ResultsIn Enshi city, HC showed the above‐normal concentrations of serum Se concentrations while RA patients were enriched in the normal range (70–150 ng mL−1) or below. RA patients with higher Se levels demonstrated milder disease and lower levels of C‐reactive protein, IL‐6, RANKL and Th17 cells. In the mouse CIA model, supranutritional Se supplementation delayed disease onset, ameliorated joint pathology and reduced CD4+CD44+RANKL+ T cells. Se supplementation could suppress RANKL expression in cultured mouse Th17 cells.ConclusionSupranutritional Se suppresses RANKL‐expressing osteoclastogenic CD4+ T cells and could be beneficial to RA, which warrants formal testing in randomised clinical trials.

Pharmacological inactivation of CDK2 inhibits MYC/BCL-XL-driven leukemia in vivo through induction of cellular senescence

ABSTRACT Deregulated expression of the MYC oncogene is a frequent event during tumorigenesis and generally correlates with aggressive disease and poor prognosis. While MYC is a potent inducer of apoptosis, it often suppresses cellular senescence, which together with apoptosis is an important barrier against tumor development. For this latter function, MYC is dependent on cyclin-dependent kinase 2 (CDK2). Here, we utilized a MYC/BCL-XL-driven mouse model of acute myeloblastic leukemia (AML) to investigate whether pharmacological inhibition of CDK2 can inhibit MYC-driven tumorigenesis through induction of senescence. Purified mouse hematopoietic stem cells transduced with MYC and BCL-XL were transplanted into lethally irradiated mice, leading to the development of massive leukemia and subsequent death 15–17 days after transplantation. Upon disease onset, mice were treated with the selective CDK2 inhibitor CVT2584 or vehicle either by daily intraperitoneal injections or continuous delivery via mini-pumps. CVT2584 treatment delayed disease onset and moderately but significantly improved survival of mice. Flow cytometry revealed a significant decrease in tumor load in the spleen, liver and bone marrow of CVT2584-treated compared to vehicle-treated mice. This was correlated with induced senescence evidenced by reduced cell proliferation, increased senescence-associated β-galactosidase activity and heterochromatin foci, expression of p19ARF and p21CIP1, and reduced phosphorylation (activation) of pRb, while very few apoptotic cells were observed. In addition, phosphorylation of MYC at Ser-62 was decreased. In summary, inhibition of CDK2 delayed MYC/BCL-XL-driven AML linked to senescence induction. Our results suggest that CDK2 is a promising target for pro-senescence cancer therapy, in particular for MYC-driven tumors, including leukemia.

Time-Restricted Feeding and Caloric Restriction Impact on Spontaneous Neoplasms in Female Mice

In older humans, multiple chronic diseases and increased life expectancy impose a disproportionate socioeconomic burden. Dietary interventions are valuable strategies for promoting healthy aging. Caloric restriction (CR) without malnutrition is a robust intervention able to delay disease onset and increase survival in model organisms. However, the impracticability of chronic CR outweighs the potential long-term benefits in humans. Time-restricted feeding (TRF), i.e. the limitation in the timing of food intake without necessarily reducing caloric intake, can protect against metabolic disorders through the synchronization of the circadian rhythm. This study compares whether limiting access to ad libitum (AL) food for a few hours per day mimics the beneficial effects of a CR diet. A large cohort of C57BL/6J female mice (n=250) was distributed into five feeding paradigms at midlife: AL, TRF for 8 hours, TRF for 4 hours, 20% CR and 20% CR fed twice a day (CRx2). Pathological analyses at death reveal a shift in fatal neoplasms toward an older age in TRF8 mice. AL mice had the highest prevalence of tumors (93%) and TRF4 had the lowest (77%). The highest tumor burden was observed in AL mice while CRx2 animals had the lowest number of neoplasms. Histiocytic sarcoma and lymphoma were the most represented malignancies, with CR mice exhibiting the highest rate of histiocytic sarcoma (75%) and the lowest rate of lymphoma (10%). These results indicate that time- and calorie-restricted feeding regimens can slow down malignant neoplasm progression and extend health span in female mice, even when started in adulthood.

BTK inhibition modulates multiple immune cell populations involved in the pathogenesis of immune mediated nephritis

Lupus nephritis (LN) is a serious end organ complication of systemic lupus erythematosus. Nephrotoxic serum nephritis (NTN) is an inducible model of LN, which utilizes passive transfer of pre-formed nephrotoxic antibodies to initiate disease. In previous studies, we demonstrated that the Bruton's tyrosine kinase inhibitor, BI-BTK-1, prevents the development of nephritis in NTN when treatment was started prior to nephrotoxic serum transfer, and reverses established proteinuria as well.We manipulated the initiation and duration of BI-BTK-1 therapy in NTN to study its delayed therapeutic effects when treatment is given later in the disease course, as well as to further understand what effect BI-BTK-1 is having to prevent initiation of nephritis with early treatment. Early treatment and remission induction each correlated with decreased inflammatory macrophages, CD4+ and CD8+ T cells, and decreased B220+ B cells. Additionally, an increased proportion of resident macrophages within the CD45+ population favored a delay of disease onset and remission induction.We also studied the cellular processes involved in reactivation of nephritis by withdrawing BI-BTK-1 treatment at different time points. Treatment cessation led to either early or later onset of renal flares inversely dependent on the initial duration of BTK inhibition, as assessed by increased proteinuria and BUN levels and worse renal pathology. These flares were associated with an increase in kidney CD45+ infiltrates, including myeloid cell populations. IL-6, CD14, and CCL2 were also increased in mice developing late flares. These analyses point to the role of macrophages as an important contributor to the pathogenesis of immune mediated nephritis, and further support the therapeutic potential of BTK inhibition in this disease and related conditions.

O16 La activación de NRF2 por el compuesto A (CPDA) protege a las células-ß de la injuria inflamatoria medida por citoquinas

Introducción: el desarrollo del proceso autoinmune durante la diabetes tipo 1 (DM1) contribuye a la insulitis; en este contexto, el estrés de las células-ß y su posterior deficiencia en la secreción de insulina preceden a los signos clínicos de la enfermedad. La hiperglucemia desencadena la producción de especies reactivas de oxígeno (ROS) mitocondriales que superan la capacidad antioxidante de las células-ß conduciendo al estrés oxidativo. El microambiente inflamatorio del islote contribuye a la activación del estrés oxidativo y del retículo endoplásmico (RE) resultando en la disfunción y muerte de las células-ß. El factor de transcripción Nrf2 regula la expresión de genes citoprotectores en respuesta al estrés oxidativo, su inducción es necesaria para la fisiología normal de la célula-ß. Hemos reportado que el CpdA regula células inmunes clave en la respuesta inmune e inflamatoria y en la insulitis, linfocitos T y células dendríticas. Tambiénobservamos que el CpdA disminuye el estrés de RE inducido por citoquinas inflamatorias (IL-1ß+IFN-g; CYT) en células-ß y que la administración in vivodel CpdA retarda la aparición de hiperglucemia en un modelo murino de diabetes autoinmune. El desarrollo de nuevos agentes con acción antiinflamatoria e inmunomoduladora y potencial protector dirigido a la señalización disfuncional de las células-ß posee interés clínico en la diabetes.Objetivos: explorar el efecto del CpdA sobre la vía de señalización de Nrf2 y el estrés oxidativo inducido por CYT en las células-ß.Materiales y métodos: como modelo experimental utilizamos la línea celular de insulinoma de rata (INS-1E). Fórmula química del CpdA: cloruro de 2-(4-acetoxifenil)-2-cloro-N-metil-etilamonio. Se utilizó un plásmido reportero (ARE-LUC) para la evaluación de la actividad transcripcional de Nrf2 (luminometría), RT-qPCR (Sybr Green) para el análisis de la expresión de ARNm, WB para el análisis de la expresión de proteínas, kit comercial basado en DCFDA para evaluar ROS (fluorometría), análisis de viabilidad celular por MTT y ELISA para insulina.

What skeletal muscle has to say in amyotrophic lateral sclerosis: Implications for therapy.

Amyotrophic lateral sclerosis (ALS) is an adult onset disorder characterized by progressive neuromuscular junction (NMJ) dismantling and degeneration of motor neurons leading to atrophy and paralysis of voluntary muscles responsible for motion and breathing. Except for a minority of patients harbouring genetic mutations, the origin of most ALS cases remains elusive. Peripheral tissues, and particularly skeletal muscle, have lately demonstrated an active contribution to disease pathology attracting a growing interest for these tissues as therapeutic targets in ALS. In this sense, molecular mechanisms essential for cell and tissue homeostasis have been shown to be deregulated in the disease. These include muscle metabolism and mitochondrial activity, RNA processing, tissue-resident stem cell function responsible for muscle regeneration, and proteostasis that regulates muscle mass in adulthood. This review aims to compile scientific evidence that demonstrates the role of skeletal muscle in ALS pathology and serves as reference for development of novel therapeutic strategies targeting this tissue to delay disease onset and progression. LINKED ARTICLES: This article is part of a themed issue on Neurochemistry in Japan. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.6/issuetoc.

BDNF-overexpressing human umbilical cord mesenchymal stem cell-derived motor neurons improve motor function and prolong survival in amyotrophic lateral sclerosis mice

ABSTRACT Objective To investigate the beneficial effect of brain-derived neurotrophic factor (BDNF) -overexpressing human umbilical cord mesenchymal stem cell (hUC-MSC)-derived motor neurons in the human Cu, Zn-superoxide dismutase1 (hSOD1)G93A amyotrophic lateral sclerosis (ALS) mice. Methods The BDNF gene was transfected into hUC-MSC-derived motor neurons by the lentivirus-mediated method. hSOD1G93A mice were assigned to the ALS, ALS/MN, and ALS/MN-BDNF groups, and intrathecally administrated phosphate-buffered saline (PBS), motor neurons, or motor neurons overexpressing BDNF, respectively. The control group included non-transgenic wild-type littermates administrated PBS. One month after transplantation, the motor function of the mice was assessed by the rotarod test, and the lumbar enlargements were then isolated to detect the expression of hSOD1 and BDNF by western blotting, and the expression of choline acetyltransferase (ChAT), homeobox protein 9 (HB9), major histocompatibility complex I (MHCI) and microtubule-associated protein-2 (MAP-2) by immunofluorescence assay. Results After transplantation, mice in the ALS/MN-BDNF and ALS/MN groups both exhibited longer latency to fall and longer survival than those in the ALS group (P < 0.01 vs. P < 0.05), and the improvement was more significant in the former than in the latter. However, cell transplantation did not delay disease onset. In the lumbar enlargements of the ALS/MN-BDNF and ALS/MN groups, the expression of hSOD1 was slightly reduced without statistical significance (P > 0.05), but the expression of BDNF, ChAT and HB9, and the co-expression of MHCI and MAP-2 were significantly greater than in the ALS group (P < 0.01), with the differences also being more prominent in the former group than in the latter. Conclusions Transplantation of BDNF-overexpressing hUC-MSC-derived motor neurons can improve motor performance and prolong the survival of hSOD1G93A mice. Combining stem cell-derived motor neurons with BDNF might provide a new therapeutic strategy for ALS.

Modulation of experimental autoimmune encephalomyelitis through colonisation of the gut with Escherichia coli.

Multiple sclerosis (MS) is a neuro-inflammatory autoimmune disease of the central nervous system (CNS) that affects young adults. It is characterised by the development of demyelinating lesions and inflammation within the CNS. Although the causes of MS are still elusive, recent work using patient samples and experimental animal models has demonstrated a strong relationship between the gut microbiota and its contribution to CNS inflammation and MS. While there is no cure for MS, alteration of the gut microbiota composition through the use of probiotics is a very promising treatment. However, while most recent works have focused on the use of probiotics to modify pre-existing disease, little is known about its role in protecting from the establishment of MS. In this study, we determined whether colonisation with the probiotic bacterium Escherichia coli strain Nissle 1917 (EcN) could be used as a prophylactic strategy to prevent or alter the development of experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS. We found that double gavage (two doses) of EcN before induction of EAE delayed disease onset and decreased disease severity. We also found that EcN-treated mice had decreased amounts of perivascular cuffing, CD4+ T cell infiltration into the CNS, together with significantly decreased absolute numbers of Th1 cells, and reduced activation of microglia. Although further studies are necessary to comprehend the exact protective mechanisms induced, our study supports a promising use of EcN as a probiotic for the prevention of MS.

Chronic administration of P2X7 receptor antagonist JNJ-47965567 delays disease onset and progression, and improves motor performance in ALS SOD1G93A female mice.

ABSTRACTNeuroinflammation is one of the main physiopathological mechanisms of amyotrophic lateral sclerosis (ALS), produced by the chronic activation of microglia in the CNS. This process is triggered by the persistent activation of the ATP-gated P2X7 receptor (P2RX7, hereafter referred to as P2X7R). The present study aimed to evaluate the effects of the chronic treatment with the P2X7R antagonist JNJ-47965567 in the development and progression of ALS in the SOD1G93A murine model. SOD1G93A mice were intraperitoneally (i.p.) injected with either 30 mg/kg of JNJ-47965567 or vehicle 4 times per week, from pre-onset age (here, postnatal day 60; P60) until study endpoint. Body weight, motor coordination, phenotypic score, disease onset and survival were measured throughout the study, and compared between vehicle- and drug-injected groups. Treatment with the P2X7R antagonist JNJ-47965567 delayed disease onset, reduced body weight loss and improved motor coordination and phenotypic score in female SOD1G93A mice, although it did not increase lifespan. Interestingly, neither beneficial nor detrimental effects were observed in males in any of the analyzed parameters. Treatment did not affect motor neuron survival or ChAT, Iba-1 and P2X7R protein expression in endpoint individuals of mixed sexes. Overall, chronic administration of JNJ-47965567 for 4 times per week to SOD1G93A mice from pre-onset stage altered disease progression in female individuals while it did not have any effect in males. Our results suggest a partial, yet important, effect of P2X7R in the development and progression of ALS.

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.