RESUMO
BACKGROUND AND PURPOSE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with limited treatment options. RNS60 is an immunomodulatory and neuroprotective investigational product that has shown efficacy in animal models of ALS and other neurodegenerative diseases. Its administration has been safe and well tolerated in ALS subjects in previous early phase trials. METHODS: This was a phase II, multicentre, randomized, double-blind, placebo-controlled, parallel-group trial. Participants diagnosed with definite, probable or probable laboratory-supported ALS were assigned to receive RNS60 or placebo administered for 24 weeks intravenously (375 ml) once a week and via nebulization (4 ml/day) on non-infusion days, followed by an additional 24 weeks off-treatment. The primary objective was to measure the effects of RNS60 treatment on selected biomarkers of inflammation and neurodegeneration in peripheral blood. Secondary objectives were to measure the effect of RNS60 on functional impairment (ALS Functional Rating Scale-Revised), a measure of self-sufficiency, respiratory function (forced vital capacity, FVC), quality of life (ALS Assessment Questionnaire-40, ALSAQ-40) and survival. Tolerability and safety were assessed. RESULTS: Seventy-four participants were assigned to RNS60 and 73 to placebo. Assessed biomarkers did not differ between arms. The mean rate of decline in FVC and the eating and drinking domain of ALSAQ-40 was slower in the RNS60 arm (FVC, difference 0.41 per week, standard error 0.16, p = 0.0101; ALSAQ-40, difference -0.19 per week, standard error 0.10, p = 0.0319). Adverse events were similar in the two arms. In a post hoc analysis, neurofilament light chain increased over time in bulbar onset placebo participants whilst remaining stable in those treated with RNS60. CONCLUSIONS: The positive effects of RNS60 on selected measures of respiratory and bulbar function warrant further investigation.
Assuntos
Esclerose Lateral Amiotrófica , Doenças Neurodegenerativas , Humanos , Esclerose Lateral Amiotrófica/diagnóstico , Qualidade de Vida , Método Duplo-Cego , Biomarcadores , Resultado do TratamentoRESUMO
Monocyte chemoattractant protein-1 (MCP1) is one of the most powerful pro-inflammatory chemokines. However, its signaling is pivotal in driving injured axon and muscle regeneration. We previously reported that MCP1 is more strongly upregulated in the nervous system of slow-progressing than fast-progressing SOD1G93A mice, the latter showing a poor immune response and eventual massive nerve and muscle degeneration. To assess the MCP1-mediated therapeutic role, we boosted the chemokine along the motor unit of the two SOD1G93A models through a single intramuscular injection of a scAAV9 vector engineered with the Mcp1 gene. We provided direct evidence underlying the pivotal role of the immune response in driving skeletal muscle regeneration and thus the speed of ALS progression. The comparative study performed in fast- and slow-progressing SOD1G93A mice spotlights the nature and temporal activation of the inflammatory response as limiting factors to preserve the periphery and interfere with the disease course. In addition, we recorded a novel pleiotropic role of MCP1 in promoting peripheral axon regeneration and modulating neuroinflammation, ultimately preventing neurodegeneration. Altogether, these observations highlight the immune response as a key determinant for disease variability and proffer a reasonable explanation for the failure of systemic immunomodulatory treatments, suggesting new potential strategies to hamper ALS progression.
Assuntos
Esclerose Lateral Amiotrófica , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/terapia , Animais , Axônios , Modelos Animais de Doenças , Imunidade , Camundongos , Camundongos Transgênicos , Músculo Esquelético , Regeneração Nervosa , Superóxido Dismutase/genética , Superóxido Dismutase-1/genéticaRESUMO
Dysfunction and degeneration of synapses is a common feature of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). A GGGGCC hexanucleotide repeat expansion in the C9ORF72 gene is the main genetic cause of ALS/FTD (C9ALS/FTD). The repeat expansion leads to reduced expression of the C9orf72 protein. How C9orf72 haploinsufficiency contributes to disease has not been resolved. Here we identify the synapsin family of synaptic vesicle proteins, the most abundant group of synaptic phosphoproteins, as novel interactors of C9orf72 at synapses and show that C9orf72 plays a cell-autonomous role in the regulation of excitatory synapses. We mapped the interaction of C9orf72 and synapsin to the N-terminal longin domain of C9orf72 and the conserved C domain of synapsin, and show interaction of the endogenous proteins in synapses. Functionally, C9orf72 deficiency reduced the number of excitatory synapses and decreased synapsin levels at remaining synapses in vitro in hippocampal neuron cultures and in vivo in the hippocampal mossy fibre system of C9orf72 knockout mice. Consistent with synaptic dysfunction, electrophysiological recordings identified impaired excitatory neurotransmission and network function in hippocampal neuron cultures with reduced C9orf72 expression, which correlated with a severe depletion of synaptic vesicles from excitatory synapses in the hippocampus of C9orf72 knockout mice. Finally, neuropathological analysis of post-mortem sections of C9ALS/FTD patient hippocampus with C9orf72 haploinsufficiency revealed a marked reduction in synapsin, indicating that disruption of the interaction between C9orf72 and synapsin may contribute to ALS/FTD pathobiology. Thus, our data show that C9orf72 plays a cell-autonomous role in the regulation of neurotransmission at excitatory synapses by interaction with synapsin and modulation of synaptic vesicle pools, and identify a novel role for C9orf72 haploinsufficiency in synaptic dysfunction in C9ALS/FTD.
Assuntos
Esclerose Lateral Amiotrófica , Proteína C9orf72/metabolismo , Demência Frontotemporal , Sinapsinas/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Proteína C9orf72/genética , Expansão das Repetições de DNA , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Camundongos , Camundongos Knockout , Sinapses/patologiaRESUMO
BACKGROUND AND AIMS: Recently, it has been hypothesized that Tri-Ponderal Mass Index (TMI) may be a valid alternative to Body Mass Index (BMI) when measuring body fat in adolescents. We aimed to verify whether TMI has better accuracy than BMI in discriminating central obesity and hypertension in adolescents with overweight. METHODS AND RESULTS: This monocentric and retrospective cross-sectional study included 3749 pupils, 1889 males and 1860 females, aged 12-13. BMI (kg/m2) was calculated and expressed as percentiles and as z-scores. TMI (kg/m3) was calculated, and we used pre-defined cut-off previously proposed by Peterson et al.. For central obesity we adopted the Waist-to-Height Ratio (WHtR) discriminatory value of 0.5. Hypertension was defined as blood pressure ≥95th percentile of age- sex-, and height-specific references recommended by NHBPEP Working Group. The discriminant ability of TMI, BMI and BMI z-score, with respect to central obesity and hypertension, was investigated using non-parametric receiver operating characteristic analysis. The overall misclassification rate for central obesity was 8.88% for TMI vs 14.10% for BMI percentiles and vs 14.92% for BMI z-scores (P < 0.001). The overall misclassification rate for hypertension was 7.50% for TMI vs 22.03% for BMI percentiles and vs 25.19% for BMI z-scores (P < 0.001). CONCLUSION: TMI is a superior body fat index and it could discriminate body fat distribution more accurately than BMI. This supports the use of TMI, in association with WHtR, to characterize adolescents with overweight and high cardio-metabolic risk. Our analysis needs to be extended to other ethnic groups and replicated in a wider age range and in longitudinal studies.
Assuntos
Adiposidade , Pressão Sanguínea , Índice de Massa Corporal , Hipertensão/diagnóstico , Obesidade Abdominal/diagnóstico , Obesidade Infantil/diagnóstico , Adolescente , Fatores Etários , Fatores de Risco Cardiometabólico , Criança , Estudos Transversais , Diagnóstico Diferencial , Feminino , Humanos , Hipertensão/epidemiologia , Hipertensão/fisiopatologia , Itália/epidemiologia , Masculino , Obesidade Abdominal/epidemiologia , Obesidade Abdominal/fisiopatologia , Obesidade Infantil/epidemiologia , Obesidade Infantil/fisiopatologia , Valor Preditivo dos Testes , Prevalência , Reprodutibilidade dos Testes , Estudos Retrospectivos , Medição de Risco , Relação Cintura-QuadrilRESUMO
Amyotrophic lateral sclerosis (ALS) is a disease with a resilient neuroinflammatory component caused by activated microglia and infiltrated immune cells. How to successfully balance neuroprotective versus neurotoxic actions through the use of anti-inflammatory agents is still under debate. There has been a boost of awareness regarding the role of extracellular ATP and purinergic receptors in modulating the physiological and pathological mechanisms in the nervous system. Particularly in ALS, it is known that the purinergic ionotropic P2X7 receptor plays a dual role in disease progression by acting at different cellular and molecular levels. In this context, we previously demonstrated that the P2X7 receptor antagonist, brilliant blue G, reduces neuroinflammation and ameliorates some of the pathological features of ALS in the SOD1-G93A mouse model. Here, we test the novel, noncommercially available, and centrally permeant Axxam proprietary P2X7 antagonist, AXX71, in SOD1-G93A mice, by assessing some behavioral and molecular parameters, among which are disease progression, survival, gliosis, and motor neuron wealth. We demonstrate that AXX71 affects the early symptomatic phase of the disease by reducing microglia-related proinflammatory markers and autophagy without affecting the anti-inflammatory markers or motor neuron survival. Our results suggest that P2X7 modulation can be further investigated as a therapeutic strategy in preclinical studies, and exploited in ALS clinical trials.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Anti-Inflamatórios/uso terapêutico , Autofagia/efeitos dos fármacos , Progressão da Doença , Antagonistas do Receptor Purinérgico P2X/uso terapêutico , Superóxido Dismutase/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Anti-Inflamatórios/farmacocinética , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Atividade Motora/efeitos dos fármacos , Força Muscular/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2X/farmacocinética , Receptores Purinérgicos P2X/metabolismoRESUMO
HLA-C expression is associated with a differential ability to control HIV-1 infection. Higher HLA-C levels may lead to better control of HIV-1 infection through both a higher efficiency of antigen presentation to cytotoxic T lymphocytes and the triggering of activating killer immunoglobulin-like receptors on NK cells, whereas lower levels may provide poor HIV-1 control and rapid progression to AIDS. We characterized the relative amounts of HLA-C heterotrimers (heavy chain/ß2 microglobulin [ß2m]/peptide) and HLA-C free heavy chains on peripheral blood mononuclear cells (PBMCs) from healthy blood donors harboring both alleles with stable or unstable binding to ß2m/peptide. We analyzed the stability of HLA-C heterotrimers of different allotypes and the infectivity of HIV-1 virions produced by PBMCs with various allotypes. We observed significant differences in HLA-C heterotrimer stability and in expression levels. We found that R5 HIV-1 virions produced by PBMCs harboring unstable HLA-C alleles were more infectious than those produced by PBMCs carrying the stable variants. We propose that HIV-1 infectivity might depend both on the amounts of HLA-C molecules and on their stability as trimeric complex. According to this model, individuals with low-expression HLA-C alleles and unstable binding to ß2m/peptide might have worse control of HIV-1 infection and an intrinsically higher capacity to support viral replication.IMPORTANCE Following HIV-1 infection, some people advance rapidly to AIDS while others have slow disease progression. HLA-C, a molecule involved in immunity, is a key determinant of HIV-1 control. Here we reveal how HLA-C variants contribute to the modulation of viral infectivity. HLA-C is present on the cell surface in two different conformations. The immunologically active conformation is part of a complex that includes ß2 microglobulin/peptide; the other conformation is not bound to ß2 microglobulin/peptide and can associate with HIV-1, increasing its infectivity. Individuals with HLA-C variants with a predominance of immunologically active conformations would display stronger immunity to HIV-1, reduced viral infectivity and effective control of HIV-1 infection, while subjects with HLA-C variants that easily dissociate from ß2 microglobulin/peptide would have a reduced immunological response to HIV-1 and produce more infectious virions. This study provides new information that could be useful in the design of novel vaccine strategies and therapeutic approaches to HIV-1.
Assuntos
Membrana Celular/imunologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Antígenos HLA-C/genética , Leucócitos Mononucleares/imunologia , Adulto , Alelos , Apresentação de Antígeno , Doadores de Sangue , Membrana Celular/genética , Membrana Celular/metabolismo , Feminino , Infecções por HIV/virologia , HIV-1/patogenicidade , Antígenos HLA-C/química , Antígenos HLA-C/imunologia , Antígenos HLA-C/metabolismo , Antígenos de Histocompatibilidade Classe I/classificação , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Células Matadoras Naturais/imunologia , Masculino , Pessoa de Meia-Idade , Linfócitos T Citotóxicos/imunologia , Adulto Jovem , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismoRESUMO
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects the motor neuromuscular system leading to complete paralysis and premature death. The multifactorial nature of ALS that involves both cell-autonomous and non-cell-autonomous processes contributes to the lack of effective therapies, usually targeted to a single pathogenic mechanism. RNS60, an experimental drug containing oxygenated nanobubbles generated by modified Taylor-Couette-Poiseuille flow with elevated oxygen pressure, has shown anti-inflammatory and neuroprotective properties in different experimental paradigms. Since RNS60 interferes with multiple cellular mechanisms known to be involved in ALS pathology, we evaluated its effect in in vitro and in vivo models of ALS. METHODS: Co-cultures of primary microglia/spinal neurons exposed to LPS and astrocytes/spinal neurons from SOD1G93A mice were used to examine the effect of RNS60 or normal saline (NS) on the selective motor neuron degeneration. Transgenic SOD1G93A mice were treated with RNS60 or NS (300 µl/mouse intraperitoneally every other day) starting at the disease onset and examined for disease progression as well as pathological and biochemical alterations. RESULTS: RNS60 protected motor neurons in in vitro paradigms and slowed the disease progression of C57BL/6-SOD1G93A mice through a significant protection of spinal motor neurons and neuromuscular junctions. This was mediated by the (i) activation of an antioxidant response and generation of an anti-inflammatory environment in the spinal cord; (ii) activation of the PI3K-Akt pro-survival pathway in the spinal cord and sciatic nerves; (iii) reduced demyelination of the sciatic nerves; and (iv) elevation of peripheral CD4+/Foxp3+ T regulatory cell numbers. RNS60 did not show the same effects in 129Sv-SOD1G93A mice, which are unable to activate a protective immune response. CONCLUSION: RNS60 demonstrated significant therapeutic efficacy in C57BL/6-SOD1G93A mice by virtue of its effects on multiple disease mechanisms in motor neurons, glial cells, and peripheral immune cells. These findings, together with the excellent clinical safety profile, make RNS60 a promising candidate for ALS therapy and support further studies to unravel its molecular mechanism of action. In addition, the differences in efficacy of RNS60 in SOD1G93A mice of different strains may be relevant for identifying potential markers to predict efficacy in clinical trials.
Assuntos
Esclerose Lateral Amiotrófica/complicações , Esclerose Lateral Amiotrófica/patologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Neuroglia/efeitos dos fármacos , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Embrião de Mamíferos , Proteína Glial Fibrilar Ácida/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Transtornos Motores/tratamento farmacológico , Transtornos Motores/etiologia , Neurônios Motores/efeitos dos fármacos , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/patologia , Crescimento Neuronal/efeitos dos fármacos , Doenças do Sistema Nervoso Periférico/etiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Cloreto de Sódio/uso terapêutico , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
BACKGROUND: Combined antiretroviral therapy has drastically reduced mortality and morbidity of HIV-infected individuals. Nevertheless long-term toxicity and appearance of viral resistance hampers the prolonged effectiveness of combination therapy, requiring a continuous input of drugs to replace those utilized in combination regimens. We here investigated the anti-HIV activity of novel derivatives of the suradista chemical class. METHODS: Compounds were tested on acute HIV-1 infection of activated peripheral blood mononuclear cells. HIV production was monitored by enzyme-linked immunosorbent assay measuring the protein p24 released in culture supernatants. Fusion assays were carried out to study the mechanism of action of these compounds. A modified version of a previously established recombinant vaccinia virus-based assay was used measuring activation of a reporter gene upon fusion of two distinct cell populations. Flow cytometry was performed in competition assays for the binding of several antibodies targeting different sites of the viral envelope glycoprotein gp120, or the receptor CD4, or the coreceptors CXCR4 and CCR5. RESULTS: Four compounds inhibited replication of a prototypic R5 (BaL) and X4 (IIIB) laboratory-adapted HIV-1 strain at low micromolar concentrations, in the absence of cytotoxicity. Approximately a ten fold greater activity was achieved against the X4 as compared to the R5 strain. The compounds blocked X4 and R5 HIV-1 fusion, a step of viral entry. This activity appeared specific for HIV-1, as entry of human herpesvirus 6 (HHV-6) and influenza virus was not substantially affected. Further investigation of the inhibitory mechanism revealed that these new molecules target the viral envelope, rather than the coreceptors, as previously shown for a congener of the same class characterized by a long plasmatic half-life. Indeed ND-4043, the most active compound, specifically competed with binding of monoclonal antibodies against the CD4-binding site (CD4-BS) and coreceptor-binding site (CoR-BS) of gp120. These compounds displayed broad anti-HIV activity, as they inhibited various primary R5, X4 and, importantly, dualtropic R5X4 HIV-1 isolates. Of the four derivatives tested, the dimeric compounds were consistently more potent than the monomeric ones. CONCLUSIONS: Given their unique features, these molecules represent promising candidates for further development and exploitation as anti-HIV therapeutics.
Assuntos
Inibidores da Fusão de HIV/farmacologia , HIV-1/fisiologia , Internalização do Vírus/efeitos dos fármacos , Células 3T3 , Animais , Antivirais/farmacologia , Benzilaminas , Morte Celular/efeitos dos fármacos , Linhagem Celular , Ciclamos , Cicloexanos/farmacologia , Citometria de Fluxo , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Compostos Heterocíclicos/farmacologia , Humanos , Maraviroc , Fusão de Membrana/efeitos dos fármacos , Camundongos , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Triazóis/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
SOD1 gene is associated with progressive motor neuron degeneration in the familiar forms of amyotrophic lateral sclerosis. Although studies on mutant human SOD1 transgenic rodent models have provided important insights into disease pathogenesis, they have not led to the discovery of early biomarkers or effective therapies in human disease. The recent generation of a transgenic swine model expressing the human pathological hSOD1G93A gene, which recapitulates the course of human disease, represents an interesting tool for the identification of early disease mechanisms and diagnostic biomarkers. Here, we analyze the activation state of CNS cells in transgenic pigs during the disease course and investigate whether changes in neuronal and glial cell activation state can be reflected by the amount of extracellular vesicles they release in biological fluids. To assess the activation state of neural cells, we performed a biochemical characterization of neurons and glial cells in the spinal cords of hSOD1G93A pigs during the disease course. Quantification of EVs of CNS cell origin was performed in cerebrospinal fluid and plasma of transgenic pigs at different disease stages by Western blot and peptide microarray analyses. We report an early activation of oligodendrocytes in hSOD1G93A transgenic tissue followed by astrocyte and microglia activation, especially in animals with motor symptoms. At late asymptomatic stage, EV production from astrocytes and microglia is increased in the cerebrospinal fluid, but not in the plasma, of transgenic pigs reflecting donor cell activation in the spinal cord. Estimation of EV production by biochemical analyses is corroborated by direct quantification of neuron- and microglia-derived EVs in the cerebrospinal fluid by a Membrane Sensing Peptide enabled on-chip analysis that provides fast results and low sample consumption. Collectively, our data indicate that alteration in astrocytic EV production precedes the onset of disease symptoms in the hSODG93A swine model, mirroring donor cell activation in the spinal cord, and suggest that EV measurements from the cells first activated in the ALS pig model, i.e. OPCs, may further improve early disease detection.
Assuntos
Esclerose Lateral Amiotrófica , Vesículas Extracelulares , Camundongos , Animais , Humanos , Suínos , Superóxido Dismutase-1/genética , Neurônios Motores/metabolismo , Superóxido Dismutase/genética , Camundongos Transgênicos , Esclerose Lateral Amiotrófica/patologia , Medula Espinal/patologia , Neuroglia/patologia , Biomarcadores/metabolismo , Peptídeos/metabolismo , Modelos Animais de DoençasRESUMO
Introduction: SARS-CoV-2 pandemic still poses a significant burden on global health and economy, especially for symptoms persisting beyond the acute disease. COVID-19 manifests with various degrees of severity and the identification of early biomarkers capable of stratifying patient based on risk of progression could allow tailored treatments. Methods: We longitudinally analyzed 67 patients, classified according to a WHO ordinal scale as having Mild, Moderate, or Severe COVID-19. Peripheral blood samples were prospectively collected at hospital admission and during a 6-month follow-up after discharge. Several subsets and markers of the innate and adaptive immunity were monitored as putative factors associated with COVID-19 symptoms. Results: More than 50 immunological parameters were associated with disease severity. A decision tree including the main clinical, laboratory, and biological variables at admission identified low NK-cell precursors and CD14+CD91+ monocytes, and high CD8+ Effector Memory T cell frequencies as the most robust immunological correlates of COVID-19 severity and reduced survival. Moreover, low regulatory B-cell frequency at one month was associated with the susceptibility to develop long COVID at six months, likely due to their immunomodulatory ability. Discussion: These results highlight the profound perturbation of the immune response during COVID-19. The evaluation of specific innate and adaptive immune-cell subsets allows to distinguish between different acute and persistent COVID-19 symptoms.
Assuntos
COVID-19 , SARS-CoV-2 , Índice de Gravidade de Doença , Humanos , COVID-19/imunologia , COVID-19/mortalidade , Masculino , Feminino , Pessoa de Meia-Idade , SARS-CoV-2/imunologia , Prognóstico , Idoso , Estudos Longitudinais , Adulto , Biomarcadores/sangue , Linfócitos T CD8-Positivos/imunologia , Imunidade Adaptativa , Células Matadoras Naturais/imunologia , Imunidade InataRESUMO
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the loss of motor neurons and neuromuscular impairment leading to complete paralysis, respiratory failure and premature death. The pathogenesis of the disease is multifactorial and noncell-autonomous involving the central and peripheral compartments of the neuromuscular axis and the skeletal muscle. Advanced clinical trials on specific ALS-related pathways have failed to significantly slow the disease. Therapy with stem cells from different sources has provided a promising strategy to protect the motor units exerting their effect through multiple mechanisms including neurotrophic support and excitotoxicity and neuroinflammation modulation, as evidenced from preclinical studies. Several phase I and II clinical trial of ALS patients have been developed showing positive effects in terms of safety and tolerability. However, the modest results on functional improvement in ALS patients suggest that only a coordinated effort between basic and clinical researchers could solve many problems, such as selecting the ideal stem cell source, identifying their mechanism of action and expected clinical outcomes. A promising approach may be stem cells selected or engineered to deliver optimal growth factor support at multiple sites along the neuromuscular pathway. This review covers recent advances in stem cell therapies in animal models of ALS, as well as detailing the human clinical trials that have been done and are currently undergoing development.
Assuntos
Esclerose Lateral Amiotrófica , Doenças Neurodegenerativas , Animais , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios Motores/metabolismo , Transplante de Células-Tronco/métodos , Modelos Animais de DoençasRESUMO
The chemokine receptor CCR5 is utilized as a critical coreceptor by most primary HIV-1 strains. While the lack of structural information on CCR5 has hampered the rational design of specific inhibitors, mimetics of the chemokines that naturally bind CCR5 can be molecularly engineered. We used a structure-guided approach to design peptide mimetics of the N-loop and ß1-strand regions of regulated on activation normal T-cell-expressed and secreted (RANTES)/CCL5, which contain the primary molecular determinants of HIV-1 blockade. Rational modifications were sequentially introduced into the N-loop/ß1-strand sequence, leading to the generation of mimetics with potent activity against a broad spectrum of CCR5-specific HIV-1 isolates (IC(50) range: 104-640 nM) but lacking activity against CXCR4-specific HIV-1 isolates. Functional enhancement was initially achieved with the stabilization of the N loop in the ß-extended conformation adopted in full-length RANTES, as confirmed by nuclear magnetic resonance (NMR) analysis. However, the most dramatic increase in antiviral potency resulted from the engraftment of an in silico-optimized linker segment designed using de novo structure-prediction algorithms to stabilize the C-terminal α-helix and experimentally validated by NMR. Our mimetics exerted CCR5-antagonistic effects, demonstrating that the antiviral and proinflammatory functions of RANTES can be uncoupled. RANTES peptide mimetics provide new leads for the development of safe and effective HIV-1 entry inhibitors.
Assuntos
Fármacos Anti-HIV/síntese química , Materiais Biomiméticos/síntese química , Antagonistas dos Receptores CCR5 , Quimiocina CCL5/síntese química , HIV-1/efeitos dos fármacos , Peptídeos/síntese química , Sequência de Aminoácidos , Fármacos Anti-HIV/farmacologia , Quimiotaxia/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Ressonância Magnética Nuclear Biomolecular , Peptídeos/farmacologia , Conformação Proteica , Engenharia de Proteínas , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacosRESUMO
Understanding how antibody responses to SARS-CoV-2 evolve during infection may provide important insight into therapeutic approaches and vaccination for COVID-19. Here we profile the antibody responses of 162 COVID-19 symptomatic patients in the COVID-BioB cohort followed longitudinally for up to eight months from symptom onset to find SARS-CoV-2 neutralization, as well as antibodies either recognizing SARS-CoV-2 spike antigens and nucleoprotein, or specific for S2 antigen of seasonal beta-coronaviruses and hemagglutinin of the H1N1 flu virus. The presence of neutralizing antibodies within the first weeks from symptoms onset correlates with time to a negative swab result (p = 0.002), while the lack of neutralizing capacity correlates with an increased risk of a fatal outcome (p = 0.008). Neutralizing antibody titers progressively drop after 5-8 weeks but are still detectable up to 8 months in the majority of recovered patients regardless of age or co-morbidities, with IgG to spike antigens providing the best correlate of neutralization. Antibody responses to seasonal coronaviruses are temporarily boosted, and parallel those to SARS-CoV-2 without dampening the specific response or worsening disease progression. Our results thus suggest compromised immune responses to the SARS-CoV-2 spike to be a major trait of COVID-19 patients with critical conditions, and thereby inform on the planning of COVID-19 patient care and therapy prioritization.
Assuntos
Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , COVID-19/mortalidade , SARS-CoV-2/imunologia , Idoso , Anticorpos Antivirais/imunologia , Formação de Anticorpos , Betacoronavirus/imunologia , COVID-19/virologia , Feminino , Humanos , Imunoglobulina G/imunologia , Cinética , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/imunologia , Taxa de SobrevidaRESUMO
BACKGROUND: Little information is available on a long-term follow-up in Bartter syndrome type I and II. METHODS: Clinical presentation, treatment and long-term follow-up (5.0-21, median 11 years) were evaluated in 15 Italian patients with homozygous (n = 7) or compound heterozygous (n = 8) mutations in the SLC12A1 (n = 10) or KCNJ1 (n = 5) genes. RESULTS: Thirteen new mutations were identified. The 15 children were born pre-term with a normal for gestational age body weight. Medical treatment at the last follow-up control included supplementation with potassium in 13, non-steroidal anti-inflammatory agents in 12 and gastroprotective drugs in five patients. At last follow-up, body weight and height were within normal ranges in the patients. Glomerular filtration rate was <90 mL/min/1.73 m(2) in four patients (one of them with a pathologically increased urinary protein excretion). In three patients, abdominal ultrasound detected gallstones. The group of patients with antenatal Bartter syndrome had a lower renin ratio (P < 0.05) and a higher standard deviation score (SDS) for height (P < 0.05) than a previously studied group of patients with classical Bartter syndrome. CONCLUSIONS: Patients with Bartter syndrome type I and II tend to present a satisfactory prognosis after a median follow-up of more than 10 years. Gallstones might represent a new complication of antenatal Bartter syndrome.
Assuntos
Síndrome de Bartter/genética , Mutação/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Simportadores de Cloreto de Sódio-Potássio/genética , Síndrome de Bartter/classificação , Síndrome de Bartter/tratamento farmacológico , Estatura , Peso Corporal , Pré-Escolar , Feminino , Seguimentos , Taxa de Filtração Glomerular , Heterozigoto , Homozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Prognóstico , Membro 1 da Família 12 de Carreador de Soluto , Fatores de TempoRESUMO
BACKGROUND: CXCL13 is a B and T lymphocyte chemokine that mediates neuroinflammation through its receptor CXCR5. This chemokine is highly expressed by motoneurons (MNs) in Amyotrophic Lateral Sclerosis (ALS) SOD1G93A (mSOD1) mice during the disease, particularly in fast-progressing mice. Accordingly, in this study, we investigated the role of this chemokine in ALS. METHODS: We used in vitro and in vivo experimental paradigms derived from ALS mice and patients to investigate the expression level and distribution of CXCL13/CXCR5 axis and its role in MN death and disease progression. Moreover, we compared the levels of CXCL13 in the CSF and serum of ALS patients and controls. FINDINGS: CXCL13 and CXCR5 are overexpressed in the spinal MNs and peripheral axons in mSOD1 mice. CXCL13 inhibition in the CNS of ALS mice resulted in the exacerbation of motor impairment (n = 4/group;Mean_Diff.=27.81) and decrease survival (n = 14_Treated:19.2 ± 1.05wks, n = 17_Controls:20.2 ± 0.6wks; 95% CI: 0.4687-1.929). This was corroborated by evidence from primary spinal cultures where the inhibition or activation of CXCL13 exacerbated or prevented the MN loss. Besides, we found that CXCL13/CXCR5 axis is overexpressed in the spinal cord MNs of ALS patients, and CXCL13 levels in the CSF discriminate ALS (n = 30) from Multiple Sclerosis (n = 16) patients with a sensitivity of 97.56%. INTERPRETATION: We hypothesise that MNs activate CXCL13 signalling to attenuate CNS inflammation and prevent the neuromuscular denervation. The low levels of CXCL13 in the CSF of ALS patients might reflect the MN dysfunction, suggesting this chemokine as a potential clinical adjunct to discriminate ALS from other neurological diseases. FUNDING: Vaccinex, Inc.; Regione Lombardia (TRANS-ALS).
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Quimiocina CXCL13/metabolismo , Neurônios Motores/metabolismo , Receptores CXCR5/metabolismo , Transdução de Sinais , Adulto , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/etiologia , Esclerose Lateral Amiotrófica/patologia , Animais , Astrócitos/metabolismo , Biomarcadores , Células Cultivadas , Quimiocina CXCL13/genética , Quimiocinas/biossíntese , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Receptores CXCR5/genética , Transdução GenéticaRESUMO
Surface expression of human leukocyte antigen (HLA)-class I molecules is critical for modulating T/natural killer lymphocytes' effector functions. Among HLA molecules, HLA-C, the most recently evolved form of class I antigens, is subjected to both transcriptional and multiple post-transcriptional regulation mechanisms affecting its cell surface expression. Among the latter a region placed in the 3' untranslated region of HLA-C transcript contains the single nucleotide polymorphism (SNP) rs67384697 "G-ins/del" that has been found to be strictly associated with surface levels of HLA-C allomorphs because of the effect on the binding site of a microRNA (Hsa-miR-148a). Higher expression of HLA-C has been proved to influence HIV-1 infection via a better control of viremia and a slower disease progression. More importantly, the analysis of SNP rs67384697 "G-ins/del" combined with the evaluation of the HLA-Bw4/-Bw6 C1/C2 supratype, as well as the killer immunoglobulin-like receptor genetic asset, has proved to be pivotal in defining the status of Elite Controllers in the Caucasian population. Here we describe a new reliable and fast method of allele-specific real-time PCR to monitor the integrity/disruption of the binding site of the microRNA Hsa-miR-148a in a high-throughput format that can be easily applied to studies involving large cohorts of individuals.
Assuntos
MicroRNAs , Polimorfismo de Nucleotídeo Único , Alelos , Sítios de Ligação , Humanos , MicroRNAs/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
An abnormal accumulation and distribution of brain iron are common to different neurodegenerative disorders, including Parkinson's disease (PD), and alteration of genes involved in iron metabolism cause neurodegeneration with brain iron accumulation. HFE participates in the regulation of iron metabolism, its mutations are primary cause of hereditary hemochromatosis and appear to be more frequent in neurodegenerative disorders such as Alzheimer's disease and amyotrophic lateral sclerosis. However, conflicting results were obtained in previous studies aimed to verify if nucleotide variations in HFE gene act as risk modifiers for PD. We used denaturing HPLC for scanning DNA sequence variations in exon 2 and 4 of HFE gene in a cohort of 475 Italian PD patients. We identified the most common H63D, C282Y and S65C, and also other 4 rare mutation types (R66H, R224W, E277K, and T281M). The allele frequency of H63D and C282Y was not statistically different from that of 2 control groups with similar mean age or of a large cohort of the same geographical area. In addition we could not find statistical differences in the clinical phenotypes of patients carrying at least one mutated HFE allele from those with the normal allele. We conclude that in the Italian population, the most common HFE mutations, H63D and C282Y are not associated with the individual risk to develop PD, nor have specific influence on the clinical features of the disease.
Assuntos
Antígenos de Histocompatibilidade Classe I/genética , Proteínas de Membrana/genética , Mutação , Doença de Parkinson/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Ácido Aspártico/genética , Cisteína/genética , Análise Mutacional de DNA , Feminino , Frequência do Gene , Genótipo , Proteína da Hemocromatose , Histidina/genética , Humanos , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/epidemiologia , Serina/genéticaRESUMO
We analysed the parkin gene in a large consecutive series (146) of unrelated early onset Parkinson's disease (onset ?40 years of age) patients. Twelve cases (8.2%) had homozygous or compound heterozygous point mutations and/or exon rearrangements, while a single mutation was found in four subjects (2.7%). We identified eight exon rearrangements and nine point mutations, two of which were novel: c.735delT (p.C212/X224) and c.815C>G (p.C238W). Genotype-phenotype correlation revealed that parkin carriers had features similar to those of non-carrier early onset Parkinson disease patients.