RESUMO
XPC deficiency is associated with mitochondrial dysfunction, increased mitochondrial H2O2 production and sensitivity to the Complex III inhibitor antimycin A (AA), through a yet unclear mechanism. We found an imbalanced expression of several proteins that participate in important mitochondrial function and increased expression and phosphorylation of the tumor suppressor p53 in Xeroderma pigmentosum complementation group C (XP-C) (XPC-null) cells compared with an isogenic line corrected in locus with wild-type XPC (XPC-wt). Interestingly, inhibition of p53 nuclear import reversed the overexpression of mitochondrial proteins, whereas AA treatment increased p53 expression more strongly in the XP-C cells. However, inhibition of p53 substantially increased XP-C cellular sensitivity to AA treatment, suggesting that p53 is a critical factor mediating the cellular response to mitochondrial stress. On the other hand, treatment with the antioxidant N-acetylcysteine increased glutathione concentration and decreased basal H2O2 production, p53 levels and sensitivity to AA treatment in the XPC-null back to the levels found in XPC-wt cells. Thus, the results suggest a critical role for mitochondrially generated H2O2 in the regulation of p53 expression, which in turn modulates XP-C sensitivity to agents that cause mitochondrial stress.
Assuntos
Proteínas de Ligação a DNA/genética , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Transformada , HumanosRESUMO
Negative feedback loops represent a regulatory mechanism that guarantees that signaling thresholds are compatible with a physiological response. Previously, we established that Lrig1 acts through this mechanism to inhibit Ret activity. However, it is unclear whether other Lrig family members play similar roles. Here, we show that Lrig1 and Lrig3 are co-expressed in Ret-positive mouse dorsal root ganglion (DRG) neurons. Lrig3, like Lrig1, interacts with Ret and inhibits GDNF/Ret signaling. Treatment of DRG neurons with GDNF ligands induces a significant increase in the expression of Lrig1 and Lrig3. Our findings show that, whereas a single deletion of either Lrig1 or Lrig3 fails to promote Ret-mediated axonal growth, haploinsufficiency of Lrig1 in Lrig3 mutants significantly potentiates Ret signaling and axonal growth of DRG neurons in response to GDNF ligands. We observe that Lrig1 and Lrig3 act redundantly to ensure proper cutaneous innervation of nonpeptidergic axons and behavioral sensitivity to cold, which correlates with a significant increase in the expression of the cold-responsive channel TrpA1. Together, our findings provide insights into the in vivo functions through which Lrig genes control morphology, connectivity and function in sensory neurons.
Assuntos
Axônios/metabolismo , Epiderme/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais/genética , Animais , Animais Recém-Nascidos , Linhagem Celular Transformada , Gânglios Espinais/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Células HEK293 , Humanos , Ligantes , Masculino , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Neurônios Motores/metabolismo , Proteínas do Tecido Nervoso/genética , Crescimento Neuronal/genética , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
Aquaporin-4 (AQP4) is the target of the specific immunoglobulin G autoantibody (AQP4-IgG) produced in patients with neuromyelitis optica spectrum disorders (NMOSD). Previous studies demonstrated that AQP4-IgG binding to astrocytic AQP4 leads to cell-destructive lesions. However, the early physiopathological events in Müller cells in the retina are poorly understood. Here, we investigated the consequences of AQP4-IgG binding to AQP4 of Müller cells, previous to the inflammatory response, on two of AQP4's key functions, cell volume regulation response (RVD) and cell proliferation, a process closely associated with changes in cell volume. Experiments were performed in a human retinal Müller cell line (MIO-M1) exposed to complement-inactivated sera from healthy volunteers or AQP4-IgG positive NMOSD patients. We evaluated AQP4 expression (immunofluorescence and western blot), water permeability coefficient, RVD, intracellular calcium levels and membrane potential changes during hypotonic shock (fluorescence videomicroscopy) and cell proliferation (cell count and BrdU incorporation). Our results showed that AQP4-IgG binding to AQP4 induces its partial internalization, leading to the decrease of the plasma membrane water permeability, a reduction of swelling-induced increase of intracellular calcium levels and the impairment of RVD in Müller cells. The loss of AQP4 from the plasma membrane induced by AQP4-IgG positive sera delayed Müller cells' proliferation rate. We propose that Müller cell dysfunction after AQP4 removal from the plasma membrane by AQP4-IgG binding could be a non-inflammatory mechanism of retinal injury in vivo, altering cell volume homeostasis and cell proliferation and consequently, contributing to the physiopathology of NMOSD.
Assuntos
Aquaporina 4/sangue , Membrana Celular/metabolismo , Células Ependimogliais/metabolismo , Imunoglobulina G/metabolismo , Neuromielite Óptica/sangue , Retina/metabolismo , Aquaporina 4/administração & dosagem , Biomarcadores/sangue , Linhagem Celular Transformada , Membrana Celular/patologia , Proliferação de Células/fisiologia , Tamanho Celular , Células Ependimogliais/patologia , Homeostase/fisiologia , Humanos , Imunoglobulina G/administração & dosagem , Neuromielite Óptica/patologia , Retina/lesões , Retina/patologiaRESUMO
The cellular prion protein (PrPC) is anchored in the plasma membrane of cells, and it is highly present in cells of brain tissue, exerting numerous cellular and cognitive functions. The present study proves the importance of PrPC in the cellular defense mechanism and metal homeostasis in astrocytes cells. Through experimental studies using cell lines of immortalized mice astrocytes (wild type and knockout for PrPC), we showed that PrPc is involved in the apoptosis cell death process by the activation of Caspase 3, downregulation of p53, and cell cycle maintenance. Metal homeostasis was determined by inductively coupled plasma mass spectrometry technique, indicating the crucial role of PrPC to lower intracellular calcium. The lowered calcium concentration and the Caspase 3 downregulation in the PrPC-null astrocytes resulted in a faster growth rate in cells, comparing with PrPC wild-type one. The presence of PrPC shows to be essential to cell death and healthy growth. In conclusion, our results show for the first time that astrocyte knockout cells for the cellular prion protein could modulate apoptosis-dependent cell death pathways.
Assuntos
Apoptose , Astrócitos/metabolismo , Caspase 3/metabolismo , Proteínas PrPC/metabolismo , Animais , Caspase 3/genética , Linhagem Celular Transformada , Ativação Enzimática , Camundongos , Proteínas PrPC/genéticaRESUMO
Vaccination still represents the most efficient and inexpensive strategy in the control of hepatitis B virus (HBV) infection. However, about 10% of the population vaccinated with the current yeast derived vaccine, consisting of the non-glycosylated form of the small envelope protein (S) of the HBV, fail to display an adequate immune response. Therefore, there is a need for the development of new vaccines with enhanced immunogenicity. On this regard, new generation vaccines containing L and preS2-containing HBV surface proteins in addition to S, have proven to be able to bypass the lack of response of the standard vaccine. In this work, we describe the development of stable recombinant CHO-K1 and HEK293 cell lines able to produce and secrete hepatitis B subviral envelope particles (HBV-SVPs) composed by the three surface proteins of the HBV. In turn, we demonstrated that these particles induced a specific humoral immune response in experimental animals and triggered the production of antibodies with the ability to recognize the binding site of HBV with the hepatocyte. Thus, these HBV-SVPs represent a promising candidate as a new generation vaccine in order to enhance the immunogenicity of the conventional yeast derived HBV vaccine.
Assuntos
Antígenos Virais/genética , Antígenos Virais/imunologia , Vírus da Hepatite B/genética , Imunidade Humoral , Proteínas do Envelope Viral , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Células CHO , Linhagem Celular Transformada , Cricetulus , Feminino , Células HEK293 , Vírus da Hepatite B/química , Vírus da Hepatite B/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Organismos Livres de Patógenos Específicos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
The growing number of oral infections caused by the Candida species are becoming harder to treat as the commonly used antibiotics become less effective. This drawback has led to the search for alternative strategies of treatment, which include the use of antifungal molecules derived from natural products. Herein, crotoxin (CTX), the main toxin of Crotalus durissus terrificus venom, was challenged against Candida tropicalis (CBS94) and Candida dubliniensis (CBS7987) strains by in vitro antimicrobial susceptibility tests. Minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), and inhibition of biofilm formation were evaluated after CTX treatment. In addition, CTX-induced cytotoxicity in HaCaT cells was assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colorimetric assay. Native CTX showed a higher antimicrobial activity (MIC = 47 µg/mL) when compared to CTX-containing mouthwash (MIC = 750 µg/mL) and nystatin (MIC = 375 µg/mL). Candida spp biofilm formation was more sensitive to both CTX and CTX-containing mouthwash (IC100 = 12 µg/mL) when compared to nystatin (IC100 > 47 µg/mL). Moreover, significant membrane permeabilization at concentrations of 1.5 and 47 µg/mL was observed. Native CTX was less cytotoxic to HaCaT cells than CTX-containing mouthwash or nystatin between 24 and 48 h. These preliminary findings highlight the potential use of CTX in the treatment of oral candidiasis caused by resistant strains.
Assuntos
Anti-Infecciosos Locais/farmacologia , Biofilmes/efeitos dos fármacos , Candida tropicalis/efeitos dos fármacos , Candida/efeitos dos fármacos , Crotoxina/farmacologia , Antissépticos Bucais/farmacologia , Anti-Infecciosos Locais/química , Anti-Infecciosos Locais/isolamento & purificação , Biofilmes/crescimento & desenvolvimento , Candida/crescimento & desenvolvimento , Candida tropicalis/crescimento & desenvolvimento , Linhagem Celular Transformada , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Crotoxina/química , Crotoxina/isolamento & purificação , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Antissépticos Bucais/química , Resultado do TratamentoRESUMO
Mesenchymal stromal cells (MSCs) are promising candidates for cell-based therapies, mainly due to their unique biological properties such as multipotency, self-renewal and trophic/immunomodulatory effects. However, clinical use has proven complex due to limitations such as high variability of MSCs preparations and high number of cells required for therapies. These challenges could be circumvented with cell immortalization through genetic manipulation, and although many studies show that such approaches are safe, little is known about changes in other biological properties and functions of MSCs. In this study, we evaluated the impact of MSCs immortalization with the TERT gene on the purinergic system, which has emerged as a key modulator in a wide variety of pathophysiological conditions. After cell immortalization, MSCs-TERT displayed similar immunophenotypic profile and differentiation potential to primary MSCs. However, analysis of gene and protein expression exposed important alterations in the purinergic signaling of in vitro cultured MSCs-TERT. Immortalized cells upregulated the CD39/NTPDase1 enzyme and downregulated CD73/NT5E and adenosine deaminase (ADA), which had a direct impact on their nucleotide/nucleoside metabolism profile. Despite these alterations, adenosine did not accumulate in the extracellular space, due to increased uptake. MSCs-TERT cells presented an impaired in vitro immunosuppressive potential, as observed in an assay of co-culture with lymphocytes. Therefore, our data suggest that MSCs-TERT have altered expression of key enzymes of the extracellular nucleotides/nucleoside control, which altered key characteristics of these cells and can potentially change their therapeutic effects in tissue engineering in regenerative medicine.
Assuntos
Adenosina/metabolismo , Terapia de Imunossupressão , Células-Tronco Mesenquimais/citologia , Telomerase/metabolismo , 5'-Nucleotidase/metabolismo , Adenosina Desaminase/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antígenos CD , Apirase , Diferenciação Celular , Linhagem Celular Transformada , Espaço Extracelular/química , Regulação da Expressão Gênica , Humanos , Células Jurkat , Ratos Wistar , Telomerase/genéticaRESUMO
Previous work by our group has shown the pro-differentiating effects of apotransferrin (aTf) on oligodendroglial cells in vivo and in vitro. Further studies showed the remyelinating effect of aTf in animal demyelination models such as hypoxia/ischemia, where the intranasal administration of human aTf provided brain neuroprotection and reduced white matter damage, neuronal loss, and astrogliosis in different brain regions. These data led us to search for a less invasive and controlled technique to deliver aTf to the CNS. To such end, we isolated extracellular vesicles (EVs) from human and mouse plasma and different neuron and glia conditioned media and characterized them based on their quality, quantity, identity, and structural integrity by western blot, dynamic light scattering, and scanning electron microscopy. All sources yielded highly pure vesicles whose size and structures were in keeping with previous literary evidence. Given that, remarkably, EVs from all sources analyzed contained Tf receptor 1 (TfR1) in their composition, we employed two passive cargo-loading strategies which rendered successful EV loading with aTf, specifically through binding to TfR1. These results unveil EVs as potential nanovehicles of aTf to be delivered into the CNS parenchyma, and pave the way for further studies into their possible clinical application in the treatment of demyelinating diseases.
Assuntos
Apoproteínas/metabolismo , Vesículas Extracelulares/metabolismo , Nanopartículas/metabolismo , Receptores da Transferrina/metabolismo , Transferrina/metabolismo , Adulto , Animais , Apoproteínas/administração & dosagem , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Nanopartículas/administração & dosagem , Ratos , Ratos Wistar , Receptores da Transferrina/administração & dosagem , Transferrina/administração & dosagemRESUMO
Dengue is the most prevalent and rapidly transmitted mosquito-borne viral disease of humans. One of the fundamental innate immune responses to viral infections includes the processing and release of pro-inflammatory cytokines such as interleukin (IL-1ß and IL-18) through the activation of inflammasome. Dengue virus stimulates the Nod-like receptor (NLRP3-specific inflammasome), however, the specific mechanism(s) by which dengue virus activates the NLRP3 inflammasome is unknown. In this study, we investigated the activation of the NLRP3 inflammasome in endothelial cells (HMEC-1) following dengue virus infection. Our results showed that dengue infection as well as the NS2A and NS2B protein expression increase the NLRP3 inflammasome activation, and further apoptosis-associated speck-like protein containing caspase recruitment domain (ASC) oligomerization, and IL-1ß secretion through caspase-1 activation. Specifically, we have demonstrated that NS2A and NS2B, two proteins of dengue virus that behave as putative viroporins, were sufficient to stimulate the NLRP3 inflammasome complex in lipopolysaccharide (LPS)-primed endothelial cells. In summary, our observations provide insight into the dengue-induced inflammatory response mechanism and highlight the importance of DENV-2 NS2A and NS2B proteins in activation of the NLRP3 inflammasome during dengue virus infection.
Assuntos
Vírus da Dengue/imunologia , Dengue/imunologia , Células Endoteliais/fisiologia , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Viroporinas/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 1/metabolismo , Linhagem Celular Transformada , Dengue/virologia , Vírus da Dengue/patogenicidade , Humanos , Imunidade Inata , Interleucina-1beta/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas Viroporinas/genética , VirulênciaRESUMO
Photodynamic therapy (PDT) with photosensitizer verteporfin is a clinically approved vascular disrupting modality that is currently in clinical trial for cancer treatment. In this study, we evaluated PDT in combination with either mTORC1 inhibitor rapamycin or mTORC1/C2 dual inhibitor AZD2014 for therapeutic enhancement in SVEC endothelial cells. Verteporfin-PDT alone induced cell apoptosis by activating the intrinsic apoptotic pathway. However, it increased the expression of anti-apoptotic protein MCL-1 and the phosphorylation of S6, a downstream molecule of mTOR signaling. In contrast, mTOR inhibitors rapamycin and AZD2014 did not induce apoptosis in SVEC cells. They suppressed MCL-1 expression and S6 phosphorylation and imposed a potent inhibition on cell proliferation. PDT in combination with mTOR inhibitors activated the intrinsic apoptotic pathway and resulted in increased apoptosis. Combination treatments also led to sustained inhibition of cell proliferation. Although AZD2014 was more effective for cell growth inhibition and PDT enhancement than rapamycin at the higher concentrations examined in the study, both inhibitors effectively enhanced PDT response, suggesting that inhibition of mTORC1 is crucial for PDT enhancement. Our results indicate that mTOR inhibitors mechanistically cooperate with PDT for enhanced cell death and sustained growth inhibition, supporting a combination approach for therapeutic enhancement.