RESUMEN
BACKGROUND: Post-kala-azar dermal leishmaniasis (PKDL), a dermal form of the disease, occurs in some visceral leishmaniasis (VL) patients following treatment. The PKDL disease mechanism is not yet clearly understood. Here we have studied the role of dermal fibroblasts in VL and PKDL disease mechanism. METHODS: Dermal fibroblasts were grown from skin biopsy explants collected from individual VL and PKDL patients and healthy controls. Fibroblasts from the third passage were subjected to RNA sequencing to analyze differentially expressed genes (DEGs). Significantly important genes were further validated by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: Transcriptome analysis of PKDL versus VL identified 516 DEGs (263 were overrepresented and 253 were underrepresented in PKDL). Among the top hub genes, MMP2, IL1B, CXCL8, IFIH1, NFKB1A, IL6, ISG15, and EGFR were underexpressed and ACTB, HSP90AA1, RAB7A, and RPS27A were overexpressed in PKDL compared to VL. CONCLUSIONS: Our data indicate that PKDL fibroblasts may present antigens through the MHC I pathway activating CD8+ T-cell mediated response, while VL fibroblasts express nuclear factor-κB (NFκB)-mediated chemokines, IL1B, IL6, and IL8, resulting in the recruitment of natural killer (NK)-cells and monocytes to the site of infection, leading to the clearance of parasite from the skin and visceralization of the disease.
Asunto(s)
Leishmania donovani , Leishmaniasis Cutánea , Leishmaniasis Visceral , Humanos , Leishmaniasis Cutánea/parasitología , Interleucina-6/genética , Expresión Génica , Perfilación de la Expresión Génica , IndiaRESUMEN
Adenosine 3',5'-cyclic monophosphate (cAMP) is a stress sensor molecule that transduces the cellular signal when Leishmania donovani moves from insect vector to mammalian host. At this stage, the parasite membrane-bound receptor adenylate cyclase predominantly produces cAMP to cope with the oxidative assault imposed by host macrophages. However, the role of soluble adenylate cyclase of L. donovani (LdHemAC) has not been investigated fully. In the present investigation, we monitored an alternative pool of cAMP, maintained by LdHemAC. The elevated cAMP effectively transmits signals by binding to Protein Kinase A (PKA) present in the cytosol and regulates antioxidant gene expression and phosphorylates several unknown PKA substrate proteins. Menadione-catalyzed production of reactive oxygen species (ROS) mimics host oxidative condition in vitro in parasites where cAMP production and PKA activity were found increased by ~1.54 ± 0.35, and ~1.78 ± 0.47-fold, respectively while expression of LdHemAC gene elevated by ~2.18 ± 0.17-fold. The LdHemAC sense these oxidants and became activated to cyclize ATP to enhance the cAMP basal level that regulates antioxidant gene expression to rescue parasites from oxidative stress. In knockdown parasites (LdHemAC-KD), the downregulated antioxidant genes expression, namely, Sod (2.30 ± 0.46), Pxn (2.73 ± 0.15), Tdr (2.7 ± 0.12), and Gss (1.57 ± 0.15) results in decreased parasite viability while in overexpressed parasites (LdHemAC-OE), the expression was upregulated by ~5.7 ± 0.35, ~2.57 ± 0.56, ~4.7 ± 0.36, and ~2.4 ± 0.83, respectively, which possibly overcomes ROS accumulation and enhances viability. Furthermore, LdHemAC-OE higher PKA activity regulates phosphorylation of substrate proteins (~56 kDs in membrane fraction and ~25 kDs in the soluble fraction). It reduced significantly when treated with inhibitors like DDA, Rp-cAMP, and H-89 and increased by ~2.1 ± 0.28-fold, respectively under oxidative conditions. The LdHemAC-KD was found less infective to RAW 264.7 macrophages and more prone to oxidative damage as compared to LdHemAC-OE and control parasites. Together, this study demonstrates mechanistic links among LdHemAC, cAMP, and PKA in parasite survival and invasion under host oxidative condition.
Asunto(s)
Adenilil Ciclasas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Leishmania donovani/enzimología , Macrófagos/fisiología , Oxidantes/farmacología , Estrés Oxidativo/fisiología , Adenilil Ciclasas/genética , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Leishmania donovani/efectos de los fármacos , Leishmania donovani/crecimiento & desarrollo , Leishmaniasis/metabolismo , Leishmaniasis/parasitología , Leishmaniasis/patología , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Ratones , Oxidación-Reducción , Fagocitosis , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
Mutations to the cholesterol transport protein apolipoprotein E (ApoE) have been identified as a major risk factor for the development of sporadic or late-onset Alzheimer's disease (AD), with the e4 allele representing an increased risk and the rare e2 allele having a reduced risk compared to the primary e3 form. The reasons behind the change in risk are not entirely understood, though ApoE4 has been connected to inflammation and toxicity in both the brain and the periphery. The goal of this study was to better understand how the ApoE isoforms (ApoE2/3/4) confer differential AD-related risk by assessing cell-specific ApoE-related neuroinflammatory and neurotoxic effects. We compared the effects of ApoE isoforms in vitro on human astrocytes, a human immortalized microglia cell line (HMC3), and the human neuroblastoma cell line SH-SY5Y. Cells were treated for 24 h with or without recombinant ApoE2, ApoE3, or ApoE4 (20 nM) and inflammation and toxicity markers assessed. Our results indicated the expression of inflammatory cytokines IL-1ß, TNFα, and IL-6 in human astrocytes was increased in response to all ApoE isoforms, with ApoE4 evoking the highest level of cytokine expression. In response to ApoE2 or ApoE3, microglial cells showed reduced levels of microglial activation markers TREM2 and Clec7a, while ApoE4 induced increased levels of both markers. ApoE2 promoted neuron survival through increased BDNF release from astrocytes. In addition, ApoE2 promoted, while ApoE4 reduced, neuronal viability. Overall, these results suggest that ApoE4 acts on cells in the brain to promote inflammation and neuronal injury and that the deleterious effects of ApoE4 on these cells may, in part, contribute to its role as a risk factor for AD.
Asunto(s)
Apolipoproteínas E/farmacología , Biomarcadores/metabolismo , Encéfalo/efectos de los fármacos , Inflamación/metabolismo , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Proteínas Recombinantes/farmacología , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Astrocitos/citología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Citocinas/metabolismo , Humanos , Inflamación/inducido químicamente , Inflamación/diagnóstico , Interleucina-1beta/metabolismo , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Protein kinase Cϵ (PKCϵ) promotes synaptic maturation and synaptogenesis via activation of synaptic growth factors such as BDNF, NGF, and IGF. However, many of the detailed mechanisms by which PKCϵ induces synaptogenesis are not fully understood. Accumulation of PSD-95 to the postsynaptic density (PSD) is known to lead to synaptic maturation and strengthening of excitatory synapses. Here we investigated the relationship between PKCϵ and PSD-95. We show that the PKCϵ activators dicyclopropanated linoleic acid methyl ester and bryostatin 1 induce phosphorylation of PSD-95 at the serine 295 residue, increase the levels of PSD-95, and enhance its membrane localization. Elimination of the serine 295 residue in PSD-95 abolished PKCϵ-induced membrane accumulation. Knockdown of either PKCϵ or JNK1 prevented PKCϵ activator-mediated membrane accumulation of PSD-95. PKCϵ directly phosphorylated PSD-95 and JNK1 in vitro Inhibiting PKCϵ, JNK, or calcium/calmodulin-dependent kinase II activity prevented the effects of PKCϵ activators on PSD-95 phosphorylation. Increase in membrane accumulation of PKCϵ and phosphorylated PSD-95 (p-PSD-95(S295)) coincided with an increased number of synapses and increased amplitudes of excitatory post-synaptic potentials (EPSPs) in adult rat hippocampal slices. Knockdown of PKCϵ also reduced the synthesis of PSD-95 and the presynaptic protein synaptophysin by 30 and 44%, respectively. Prolonged activation of PKCϵ increased synapse number by 2-fold, increased presynaptic vesicle density, and greatly increased PSD-95 clustering. These results indicate that PKCϵ promotes synaptogenesis by activating PSD-95 phosphorylation directly through JNK1 and calcium/calmodulin-dependent kinase II and also by inducing expression of PSD-95 and synaptophysin.
Asunto(s)
Hipocampo/metabolismo , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Proteínas de la Membrana/biosíntesis , Proteína Quinasa C-epsilon/metabolismo , Membranas Sinápticas/metabolismo , Animales , Brioestatinas/farmacología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Homólogo 4 de la Proteína Discs Large , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Proteína Quinasa 8 Activada por Mitógenos/genética , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Proteína Quinasa C-epsilon/genética , Ratas , Membranas Sinápticas/genética , Sinaptofisina/biosíntesis , Sinaptofisina/genéticaRESUMEN
Apolipoprotein E4 (ApoE4) is a major genetic risk factor for several neurodegenerative disorders, including Alzheimer's disease (AD). Epigenetic dysregulation, including aberrations in histone acetylation, is also associated with AD. We show here for the first time that ApoE4 increases nuclear translocation of histone deacetylases (HDACs) in human neurons, thereby reducing BDNF expression, whereas ApoE3 increases histone 3 acetylation and upregulates BDNF expression. Amyloid-ß (Aß) oligomers, which have been implicated in AD, caused effects similar to ApoE4. Blocking low-density lipoprotein receptor-related protein 1 (LRP-1) receptor with receptor-associated protein (RAP) or LRP-1 siRNA abolished the ApoE effects. ApoE3 also induced expression of protein kinase C ε (PKCε) and PKCε retained HDACs in the cytosol. PKCε activation and ApoE3 supplementation prevented ApoE4-mediated BDNF downregulation. PKCε activation also reversed Aß oligomer- and ApoE4-induced nuclear import of HDACs, preventing the loss in BDNF. ApoE4 induced HDAC6-BDNF promoter IV binding, which reduced BDNF exon IV expression. Nuclear HDAC4 and HDAC6 were more abundant in the hippocampus of ApoE4 transgenic mice than in ApoE3 transgenic mice or wild-type controls. Nuclear translocation of HDA6 was also elevated in the hippocampus of AD patients compared with age-matched controls. These results provide new insight into the cause of synaptic loss that is the most important pathologic correlate of cognitive deficits in AD.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Apolipoproteínas E/farmacología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Encéfalo/patología , Nucléolo Celular/metabolismo , Histona Desacetilasas/metabolismo , Neuronas/ultraestructura , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Animales , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Estudios de Casos y Controles , Línea Celular Tumoral , Nucléolo Celular/efectos de los fármacos , Células Cultivadas , Colesterol/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Neuroblastoma/patología , Neuronas/efectos de los fármacos , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Transporte de Proteínas/efectos de los fármacos , Interferencia de ARN/fisiologíaRESUMEN
Visceral leishmaniasis (VL) is a tropical disease that causes severe public health problems in humans when untreated. As no licensed vaccine exists against VL, we aimed to formulate a potential MHC-restricted chimeric vaccine construct against this dreadful parasitic disease. Amastin-like protein derived from L. donovani is considered to be stable, immunogenic and non-allergic. A comprehensive established framework was used to explore the set of immunogenic epitopes with estimated population coverage of 96.08% worldwide. The rigorous assessment revealed 6 promiscuous T-epitopes which can plausibly be presented by more than 66 diverse HLA alleles. Further docking and simulation study of peptide receptor complexes identified a strong and stable binding interaction with better structural compactness. The predicted epitopes were combined with appropriate linkers and adjuvant molecules and their translation efficiency was evaluated in pET28+(a), an bacterial expression vector using in-silico cloning. Molecular docking followed by MD simulation study revealed a stable interaction between chimeric vaccine construct with TLRs. Immune simulation of the chimeric vaccine constructs showed an elevated Th1 immune response against both B and T epitopes. With this, the detailed computational analysis suggested that the chimeric vaccine construct can evoke a robust immune response against Leishmania donovani infection. Future studies are required to validate the role of amastin as a promising vaccine target.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Leishmania donovani , Leishmaniasis Visceral , Vacunas , Humanos , Leishmania donovani/genética , Epítopos , Simulación del Acoplamiento Molecular , Vacunología , Leishmaniasis Visceral/parasitología , Epítopos de Linfocito T , Epítopos de Linfocito B , Vacunas de Subunidad , Biología ComputacionalRESUMEN
Synaptic loss is the earliest pathological change in Alzheimer disease (AD) and is the pathological change most directly correlated with the degree of dementia. ApoE4 is the major genetic risk factor for the age-dependent form of AD, which accounts for 95% of cases. Here we show that in synaptic networks formed from primary hippocampal neurons in culture, apoE3, but not apoE4, prevents the loss of synaptic networks produced by amyloid ß oligomers (amylospheroids). Specific activators of PKCε, such as 8-(2-(2-pentyl-cyclopropylmethyl)-cyclopropyl)-octanoic acid methyl ester and bryostatin 1, protected against synaptic loss by amylospheroids, whereas PKCε inhibitors blocked this synaptic protection and also blocked the protection by apoE3. Blocking LRP1, an apoE receptor on the neuronal membrane, also blocked the protection by apoE. ApoE3, but not apoE4, induced the synthesis of PKCε mRNA and expression of the PKCε protein. Amyloid ß specifically blocked the expression of PKCε but had no effect on other isoforms. These results suggest that protection against synaptic loss by apoE is mediated by a novel intracellular PKCε pathway. This apoE pathway may account for much of the protective effect of apoE and reduced risk for the age-dependent form of AD. This finding supports the potential efficacy of newly developed therapeutics for AD.
Asunto(s)
Apolipoproteína E3/farmacología , Apolipoproteína E4/farmacología , Neuronas/efectos de los fármacos , Proteína Quinasa C-epsilon/metabolismo , Sinapsis/efectos de los fármacos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/farmacología , Animales , Apolipoproteína E3/genética , Apolipoproteína E3/metabolismo , Western Blotting , Brioestatinas/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colesterol/farmacología , Activación Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Neuronas/metabolismo , Neuronas/patología , Proteína Quinasa C-epsilon/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sinapsis/metabolismo , Sinapsis/patologíaRESUMEN
Protein kinase C (PKC) ε and α activation has been implicated in synaptogenesis. We used aged rats to test whether the PKCε/α activator bryostatin and PKCε-specific activator DCP-LA combined with spatial memory training could restore mushroom dendritic spinogenesis and synaptogenesis. Compared with young rats, aged, learning-impaired rats had lower memory retention; lower densities of mushroom spines and synapses in the apical dendrites of CA1 pyramidal neurons; fewer PKCε-containing presynaptic axonal boutons; and lower activation and expression of two PKCε/α substrates, the mRNA-stabilizing protein HuD and brain-derived neurotrophic factor (BDNF). PKC activator treatment combined with spatial memory training restored mushroom spines and mushroom spine synapses; rescued PKCε/α expression and PKC/HuD/BDNF signaling; and normalized memory to the levels seen in young rats. These effects were produced by treatment with either bryostatin or the PKCε-specific activator, DCP-LA. Bryostatin also reversed alterations in GABAergic inhibitory postsynaptic currents (IPSPs) in aged, learning-impaired rats. Thus, our results support the therapeutic potential of PKC activators when added to cognitive rehabilitation for inducing mushroom spine synaptogenesis and reversing memory decline associated with aging.
Asunto(s)
Envejecimiento , Dendritas/fisiología , Hipocampo/citología , Memoria/fisiología , Proteína Quinasa C/metabolismo , Sinapsis/fisiología , Anestésicos Locales/farmacología , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Brioestatinas/farmacología , Caprilatos/farmacología , Dendritas/efectos de los fármacos , Dendritas/ultraestructura , Proteínas ELAV/genética , Proteínas ELAV/metabolismo , Proteína 4 Similar a ELAV , Estimulación Eléctrica , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Lidocaína/análogos & derivados , Lidocaína/farmacología , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Memoria/efectos de los fármacos , Técnicas de Placa-Clamp , Células Piramidales/citología , Células Piramidales/efectos de los fármacos , Ratas , Sinapsis/efectos de los fármacos , Factores de TiempoRESUMEN
Chemically induced crosslinked enhanced emission (CEE) of urea and citric acid-derived carbon polymer dot (CPD) nanoparticles is established here with a rare zero linker approach, i.e. without the use of any separate crosslinkers. Such chemical CEE like any chemical reaction was achieved through amide bond formation using carbodiimide chemistry, pointing towards the feasibility of developing a general methodology for their formation through engineering the nanoparticle surface functionality. Exhaustive characterization was done to pinpoint the structure, morphology, and photophysics of the CPDs and concurrently eliminate the possibility of the involvement and interference by molecular fluorophores for the unique optical tuning of the CPDs. The structure-photophysics relation was further restated through theoretical studies involving density functional theory (DFT) that correlated significantly well with the experimental findings. Most interestingly, the CPDs revealed pH responsiveness due to the formation or hydrolysis of amide bonds with acid or base, respectively, which was manifested through a spectacular change in fluorescence emission visible to the naked eye through UV illumination. This distinct pH-dependent photoluminescence properties of CPDs opens up an enormous opportunity for interesting applications, including discriminating normal and cancerous cells, which we demonstrate herein as a proof of concept through in vitro imaging.
Asunto(s)
Neoplasias , Polímeros , Polímeros/química , Carbono/química , Fluorescencia , Colorantes Fluorescentes/química , Concentración de Iones de Hidrógeno , Neoplasias/diagnóstico por imagenRESUMEN
Conversion of biomass into nanoparticles for meaningful biomedical applications is a formidable proposition with excellent prospects but fewer patrons. A lack of general methodology for upscaled production and limited versatility of those nanoparticles are the main drawbacks. Herein, we report the creation of a DNA nanoparticle (DNA Dots) from onion genomic DNA (gDNA), a plant biomass source, through controlled hydrothermal pyrolysis in water without any chemicals. The DNA Dots are further formulated into a stimuli-responsive hydrogel through hybridization-mediated self-assembly with untransformed precursor gDNA. The versatility of the DNA Dots is recognized through its crosslinking ability with gDNA through its dangling DNA strands on the surface resulting from incomplete carbonization during annealing without the need for any external organic, inorganic, or polymeric crosslinkers. The gDNA-DNA Dots hybrid hydrogel is shown to be an excellent drug delivery vehicle for sustained release trackable through the inherent fluorescence of the DNA Dots. Interestingly, the DNA Dots are photoexcited with normal visible light to generate on-demand reactive oxygen species, making them exciting candidates for combination therapeutics. Most importantly, the ease with which the hydrogel is internalized in fibroblast cells with minimal cytotoxicity should encourage the nanotization of biomass as a tool for interesting sustainable biomedical applications.
Asunto(s)
Hidrogeles , Nanopartículas , Biomasa , Sistemas de Liberación de Medicamentos/métodos , ADNRESUMEN
Malaria is still a complex and lethal parasitic infectious disease, despite the availability of effective antimalarial drugs. Resistance of malaria parasites to current treatments necessitates new antimalarials targeting P. falciparum proteins. The present study reported the design and synthesis of a series of a 2-(4-substituted piperazin-1-yl)-N-(5-((naphthalen-2-yloxy)methyl)-1,3,4-thiadiazol-2-yl)acetamide hybrids for the inhibition of Plasmodium falciparum dihydrofolate reductase (PfDHFR) using computational biology tools followed by chemical synthesis, structural characterization, and functional analysis. The synthesized compounds were evaluated for their in vitro antimalarial activity against CQ-sensitive PfNF54 and CQ-resistant PfW2 strain. Compounds T5 and T6 are the most active compounds having anti-plasmodial activity against PfNF54 with IC50 values of 0.94 and 3.46 µM respectively. Compound T8 is the most active against the PfW2 strain having an IC50 of 3.91 µM. Further, these active hybrids (T5, T6, and T8) were also evaluated for enzyme inhibition assay against PfDHFR. All the tested compounds were non-toxic against the Hek293 cell line with good selectivity indices. Hemolysis assay also showed non-toxicity of these compounds on normal uninfected human RBCs. In silico molecular docking studies were carried out in the binding pocket of both the wild-type and quadruple mutant Pf-DHFR-TS to gain further insights into probable modes of action of active compounds. ADME prediction and physiochemical properties support their drug-likeness. Additionally, they were screened for antileishmanial activity against L. donovani promastigotes to explore broader applications. Thus, this study provides molecular frameworks for developing potent antimalarials and antileishmanial agents.
RESUMEN
Visceral leishmaniasis, which is caused by Leishmania donovani, is one of the major health problems of the Indian subcontinent. Infected hosts have been reported to have impaired lymphoproliferation. However, the fate of anergic cells is still elusive. In the present investigation, L. donovani-infected hamsters were used to study the mechanism of lymphocyte cell death. Lymph node-derived lymphocytes were analysed for apoptotic death through mitochondrial abnormality, caspase activity and DNA degradation. The data demonstrate that the disease progression leads to a gradual impairment of lymphocyte proliferation in the presence of Concanavalin A. The fate of the anergic lymphocytes is intrinsic apoptosis, which is evident by the depolarization of the mitochondrial membrane potential, cytosolic release of cytochrome c, caspase activation and DNA fragmentation. Tumour growth factor (TGF)-ß, which is secreted by macrophages, was significantly upregulated in the lymph node compartment of infected hamsters. Adding a neutralizing TGF-ß antibody and a recombinant TGF-ß resulted in the downregulation and induction of lymphocyte apoptosis, respectively. Furthermore, it has been observed that TGF-ß triggers the apoptotic death of lymphocytes through the upregulation of tyrosine phosphatase activity and that the use of sodium orthovanadate (Na(3)VO(4), a tyrosine phosphatase inhibitor) reduces the apoptotic frequency. Thus, this study clearly reports the novel involvement of tyrosine phosphatases in TGF-ß-induced lymphocyte apoptosis in Leishmania-infected hamsters.
Asunto(s)
Apoptosis , Regulación Enzimológica de la Expresión Génica , Leishmaniasis Visceral/fisiopatología , Linfocitos/inmunología , Linfocitos/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Factor de Crecimiento Transformador beta , Animales , Anticuerpos Neutralizantes/farmacología , Apoptosis/efectos de los fármacos , Cricetinae , Inhibidores Enzimáticos/farmacología , Femenino , Regulación Enzimológica de la Expresión Génica/inmunología , Leishmania donovani/inmunología , Leishmaniasis Visceral/enzimología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Activación de Linfocitos/inmunología , Mitocondrias/inmunología , Mitocondrias/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunología , Regulación hacia Arriba/inmunología , Vanadatos/farmacologíaRESUMEN
Background: Vascular risk factors such as atherosclerosis, diabetes, and elevated homocysteine levels are strongly correlated with onset of Alzheimer's disease (AD). Emerging evidence indicates that blood coagulation protein thrombin is associated with vascular and non-vascular risk factors of AD. Here, we examined the effect of thrombin and its direct inhibitor dabigatran on key mediators of neuro-inflammation and AD pathology in the retinoic acid (RA)-differentiated human neuroblastoma cell line SH-SY5Y. Methods: SH-SY5Y cells exposed to thrombin concentrations (10-100 nM) +/- 250 nM dabigatran for 24 h were analyzed for protein and gene expression. Electrophoretic mobility shift assay (EMSA) was used to determine DNA binding of NFkB. Western blotting, qRT-PCR and ELISA were used to measure the protein, mRNA, and activity levels of known AD hallmarks and signaling molecules. Results: Dabigatran treatment attenuated thrombin-induced increase in DNA binding of NFκB by 175% at 50 nM and by 77% at 100 nM thrombin concentration. Thrombin also augmented accumulation of Aß protein expression and phosphorylation of p38 MAPK, a downstream molecule in the signaling cascade, expression of pro-apoptotic mediator caspase 3, APP, tTau and pTau. Additionally, thrombin increased BACE1 activity, GSK3ß expression, and APP, BACE1, Tau and GSK3ß mRNA levels. Co-incubation with dabigatran attenuated thrombin-induced increases in the protein, mRNA, and activities of the aforesaid molecules to various extents (between -31% and -283%). Conclusion: Our data demonstrates that thrombin promotes AD-related pathological changes in neuronal cultures and suggests that use of direct oral anticoagulants may provide a therapeutic benefit against thrombin-driven neuroinflammation and downstream pathology in AD.
RESUMEN
Genetic variations in the host TLRs genes play an important role in susceptibility and/or resistance to visceral leishmaniasis by altering the host-pathogen interaction. In this study, we investigated the association between polymorphisms of TLR4 (Asp299Gly, Thr399Ile) and TLR-9 (T-1237C), with susceptibility to visceral leishmaniasis. A bi-directional PCR amplification of specific alleles technique was used to characterize the distribution of TLR4 (Asp299Gly and Thr399Ile) and TLR9 (T-1237C) polymorphisms. A total of 60 samples were randomly selected from confirmed visceral leishmaniasis patients and 24 endemic healthy volunteers. The samples were genotyped and allele frequencies were determined. We observed that TLR4 Asp299Gly and Thr399Ile genotypes were more frequent in visceral leishmaniasis patients (10% and 15% respectively) compared to controls (4.2% and 8.3% respectively). However, the differences were not significant in TLR4 Asp299Gly and Thr399Ile alleles and genotypes. In the case of TLR9, we observed the frequency of T1237C genotype was higher in visceral leishmaniasis patients (43.3%) than in healthy controls (33.3%). Statistically significant differences were observed in TLR9 T1237C alleles and genotypes. We concluded that TLR9 T1237C, but not TLR4, gene polymorphisms can be regarded as contributors to visceral leishmaniasis susceptibility among the Indian population of Bihar state.
Asunto(s)
Genotipo , Leishmania donovani/fisiología , Leishmaniasis Visceral/inmunología , Grupos de Población , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 9/metabolismo , Frecuencia de los Genes , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , India , Leishmaniasis Visceral/genética , Enfermedades Desatendidas , Polimorfismo Genético , Receptor Toll-Like 4/genética , Receptor Toll-Like 9/genéticaRESUMEN
BACKGROUND: Diabetes is one of the strongest disease-related risk factors for Alzheimer's disease (AD). In diabetics, hyperglycemia-induced microvascular complications are the major cause of end-organ injury, contributing to morbidity and mortality. Microvascular pathology is also an important and early feature of AD. The cerebral microvasculature may be a point of convergence of both diseases. Several lines of evidence also implicate thrombin in AD as well as in diabetes. OBJECTIVE: Our objective was to investigate the role of thrombin in glucose-induced brain microvascular endothelial injury. METHODS: Cultured Human brain microvascular endothelial cells (HBMVECs) were treated with 30âmM glucose±100ânM thrombin and±250ânM Dabigatran or inhibitors of PAR1, p38MAPK, MMP2, or MMP9. Cytotoxicity and thrombin activity assays on supernatants and western blotting for protein expression in lysates were performed. RESULTS: reatment of HBMVECs with 30âmM glucose increased thrombin activity and expression of inflammatory proteins TNFα, IL-6, and MMPs 2 and 9; this elevation was reduced by the thrombin inhibitor dabigatran. Direct treatment of brain endothelial cells with thrombin upregulated p38MAPK and CREB, and induced TNFα, IL6, MMP2, and MMP9 as well as oxidative stress proteins NOX4 and iNOS. Inhibition of thrombin, thrombin receptor PAR1 or p38MAPK decrease expression of inflammatory and oxidative stress proteins, implying that thrombin may play a central role in glucose-induced endothelial injury. CONCLUSION: Since preventing brain endothelial injury would preserve blood-brain barrier integrity, prevent neuroinflammation, and retain intact functioning of the neurovascular unit, inhibiting thrombin, or its downstream signaling effectors, could be a therapeutic strategy for mitigating diabetes-induced dementia.
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Antitrombinas/farmacología , Encéfalo/irrigación sanguínea , Dabigatrán/farmacología , Células Endoteliales/metabolismo , Endotelio Vascular/fisiopatología , Glucosa/toxicidad , Trombina/metabolismo , Enfermedad de Alzheimer/epidemiología , Enfermedad de Alzheimer/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Diabetes Mellitus Tipo 2/epidemiología , Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Humanos , Inflamación , Interleucina-6/metabolismo , Metaloproteinasa 2 de la Matriz/efectos de los fármacos , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/metabolismo , Microvasos/citología , NADPH Oxidasa 4/efectos de los fármacos , NADPH Oxidasa 4/metabolismo , Óxido Nítrico Sintasa de Tipo II/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Estrés Oxidativo/efectos de los fármacos , Trombina/efectos de los fármacos , Trombina/farmacología , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Leishmania secretes over 151 proteins during in vitro cultivation. Cellular functions of one such novel protein: mevalonate kinase is discussed here; signifying its importance in Leishmania infection. Visceral Leishmaniasis is a persistent infection, caused by Leishmania donovani in Indian subcontinent. This persistence is partly due to phagocytosis and evasion of host immune response. The underlying mechanism involves secretory proteins of Leishmania parasite; however, related studies are meagre. We have identified a novel secretory Leishmania donovani glycoprotein, Mevalonate kinase (MVK), and shown its importance in parasite internalization and immuno-modulation. In our studies, MVK was found to be secreted maximum after 1 h temperature stress at 37°C. Its secretion was increased by 6.5-fold in phagolysosome-like condition (pH ~5.5, 37°C) than at pH ~7.4 and 25°C. Treatment with MVK modulated host immune system by inducing interleukin-10 and interleukin-4 secretion, suppressing host's ability to kill the parasite. Peripheral blood mononuclear cell (PBMC)-derived macrophages infected with mevalonate kinase-overexpressing parasites showed an increase in intracellular parasite burden in comparison to infection with vector control parasites. Mechanism behind the increase in phagocytosis and immunosuppression was found to be phosphorylation of mitogen-activated protein (MAP) kinase pathway protein, Extracellular signal-regulated kinases-1/2, and actin scaffold protein, cortactin. Thus, we conclude that Leishmania donovani Mevalonate kinase aids in parasite engulfment and subvert the immune system by interfering with signal transduction pathways in host cells, which causes suppression of the protective response and facilitates their persistence in the host. Our work elucidates the involvement of Leishmania in the process of phagocytosis which is thought to be dependent largely on macrophages and contributes towards better understanding of host pathogen interactions.
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Leishmania donovani , Leishmaniasis Visceral , Humanos , Leucocitos Mononucleares , Fagocitosis , Fosfotransferasas (Aceptor de Grupo Alcohol)RESUMEN
Leishmaniasis comprises of a wide variety of diseases, caused by protozoan parasite belonging to the genus Leishmania. Leishmania parasites undergo different types of stress during their lifetime and have developed strategies to overcome this damage. Identifying the mechanistic approach used by the parasite in dealing with the stress is of immense importance for unfolding the survival strategy adopted by the parasite. Mevalonate kinase (MVK) is an important regulatory factor in the mevalonate pathway in both bacteria and eukaryotes. In this study, we explored the role of Leishmania donovani mevalonate kinase (LdMVK) in parasite survival under stress condition. Hydrogen peroxide (H2O2) and menadione, the two known oxidants were used to carry out the experiments. The MVK expression was found to be up regulated â¼2.1 fold and â¼2.3 fold under oxidative stress condition and under the effect of anti-Leishmania drug, AmBisome respectively. The cell viability declined under the effect of MVK inhibitor viz: vanadyl sulfate (VS). The level of intracellular ROS was also found to be increased under the effect of MVK inhibitor. To confirm the findings, LdMVK over expression (LdMVK OE) and LdMVK knockdown (LdMVK KD) parasites were generated. The level of ergosterol, an important component of plasma membrane in L. donovani, was observed and found to be reduced by nearly 60 % in LdMVK KD parasite and increased by nearly 30 % in LdMVK OE parasites as compared to wild type. However, the ergosterol content was found to be elevated under oxidative stress. Furthermore, LdMVK was also found to be associated with maintaining the plasma membrane integrity and also in preventing the peroxidation of cellular lipids when exposed to oxidative stress. The above data clearly suggests that MVK has a vital role in protecting the parasite from oxidative stress. These findings may also explore the contribution of LdMVK in drug unresponsiveness which may help in future rational drug designing for leishmaniasis.
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Ergosterol , Leishmania donovani , Estrés Oxidativo , Fosfotransferasas (Aceptor de Grupo Alcohol) , Animales , Ergosterol/biosíntesis , Peróxido de Hidrógeno/toxicidad , Leishmania donovani/enzimología , Leishmania donovani/metabolismo , Estrés Oxidativo/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismoRESUMEN
Programmed cell death (PCD) is an essential process in the growth and development of multicellular organisms. However, accumulating evidence indicates that unicellular eukaryotes can also undergo PCD with apoptosis-like features. This study demonstrates that after exposure to 0.8 mM H(2)O(2) for 9 h Entamoeba histolytica presents morphological and biochemical evidence of apoptosis-like death. Morphological characteristics of apoptosis-like death including DNA fragmentation, increased vacuolization, nuclear condensation and cell rounding were observed for H(2)O(2)-exposed trophozoites with preservation of membrane integrity. Biochemical alteration in ion fluxes is also a key feature in PCD, and H(2)O(2)-exposed trophozoites showed overproduction of reactive oxygen species, increased cytosolic Ca(2+) and decreased intracellular pH. Phosphatidylserine was also found to be expressed in the outer leaflet of the plasma membrane of the H(2)O(2)-treated trophozoites. Pretreatment with the cysteine protease inhibitor E-64d, the extracellular and intracellular Ca(2+) chelators EGTA and BAPTA/AM, and the Ca(2+) influx inhibitor verapamil prior to H(2)O(2) exposure abolished DNA fragmentation. The oxidatively stressed trophozoites also showed an increased calpain activity, indicating involvement of Ca(2+)-dependent calpain-like cysteine proteases in PCD of E. histolytica. A homogeneous caspase assay showed no significant caspase activity, and administration of caspase 1 inhibitor also did not prevent the death phenotype for the oxidatively stressed trophozoites, indicating a caspase-independent apoptosis-like death. Our observations clearly demonstrate that there is a distinct calpain-dependent but caspase-independent pathway for apoptosis-like death in oxidatively stressed E. histolytica trophozoites.
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Apoptosis/efectos de los fármacos , Entamoeba histolytica/citología , Entamoeba histolytica/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Fragmentación del ADN/efectos de los fármacos , Entamoeba histolytica/crecimiento & desarrollo , Entamoeba histolytica/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Trofozoítos/citología , Trofozoítos/efectos de los fármacos , Trofozoítos/crecimiento & desarrollo , Trofozoítos/metabolismoRESUMEN
MicroRNAs are small ribonucleic acid that act as an important regulator of gene expression at the molecular level. However, there is no comparative data on the regulation of microRNAs (miRNAs) in visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL). In this current study, we compared the expression miRNA profile in host cells (GTHP), with VL strain (GVL) and PKDL strain-infected host cell (GPKDL). Normalized read count comparison between different conditions revealed that the miRNAs are indeed differentially expressed. In GPKDL with respect to GVL and GTHP, a total of 798 and 879 miRNAs were identified, out of which 349 and 518 are known miRNAs, respectively. Comparative analysis of changes in miRNA expression suggested that the involvement of differentially expressed miRNAs in various biological processes like PI3K pathway activation, cell cycle regulation, immunomodulation, apoptosis inhibition, different cytokine production, T-cell phenotypic transitions calcium regulation, and so on. A pathway enrichment study using in silico predicted gene targets of differentially expressed miRNAs showed evidence of potentially universal immune signaling pathway effects. Whereas cytokine-cytokine receptor interaction, phagocytosis, and transforming growth factor beta (TGF-ß) signaling pathways were more highly enriched using targets of miRNAs upregulated in GPKDL. These findings could contribute to a better understanding of PKDL pathogenesis. Furthermore, the identified miRNAs could also be used as biomarkers in diagnosis, prognosis, and therapeutics of PKDL infection control.
RESUMEN
Proteins that regulate the coagulation cascade, including thrombin, are elevated in the brains of Alzheimer's disease (AD) patients. While studies using amyloid-based AD transgenic mouse models have implicated thrombin as a protein of interest, the role of thrombin in tau-based animal models has not been explored. The current study aims to determine how inhibiting thrombin could alter oxidative stress, inflammation, and AD-related proteins in a tau-based mouse model, the Tg4510. Aged Tg4510 mice were treated with the direct thrombin inhibitor dabigatran or vehicle for 7 days, brains collected, and western blot and data-independent proteomics using mass spectrometry with SWATH-MS acquisition performed to evaluate proteins related to oxidative stress, intracellular signaling, inflammation, and AD pathology. Dabigatran reduced iNOS, NOX4, and phosphorylation of tau (S396, S416). Additionally, dabigatran treatment increased expression of several signaling proteins related to cell survival and synaptic function. Increasing evidence supports a chronic procoagulant state in AD, highlighting a possible pathogenic role for thrombin. Our data demonstrate that inhibiting thrombin produces alterations in the expression of proteins involved in oxidative stress, inflammation, and AD-related pathology, suggesting that thrombin-mediated signaling affects multiple AD-related pathways providing a potential future therapeutic target.