RESUMO
Prenatal exposure to metabolism-disrupting chemicals (MDCs) has been linked to birth weight, but the molecular mechanisms remain largely unknown. In this study, we investigated gene expressions and biological pathways underlying the associations between MDCs and birth weight, using microarray transcriptomics, in a Belgian birth cohort. Whole cord blood measurements of dichlorodiphenyldichloroethylene (p,p'-DDE), polychlorinated biphenyls 153 (PCB-153), perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), and transcriptome profiling were conducted in 192 mother-child pairs. A workflow including a transcriptome-wide association study, pathway enrichment analysis with a meet-in-the-middle approach, and mediation analysis was performed to characterize the biological pathways and intermediate gene expressions of the MDC-birth weight relationship. Among 26,170 transcriptomic features, we successfully annotated five overlapping metabolism-related gene expressions associated with both an MDC and birth weight, comprising BCAT2, IVD, SLC25a16, HAS3, and MBOAT2. We found 11 overlapping pathways, and they are mostly related to genetic information processing. We found no evidence of any significant mediating effect. In conclusion, this exploratory study provides insights into transcriptome perturbations that may be involved in MDC-induced altered birth weight.
Assuntos
Poluentes Ambientais , Efeitos Tardios da Exposição Pré-Natal , Gravidez , Feminino , Humanos , Peso ao Nascer/genética , Bélgica , Transcriptoma , Coorte de Nascimento , Sangue Fetal/química , Diclorodifenil Dicloroetileno , Exposição Materna/efeitos adversos , Poluentes Ambientais/análise , Autoantígenos/análise , Proteínas de Membrana Transportadoras/análiseRESUMO
BACKGROUND: There have been few studies regarding viral involvement in the pathogenesis of renal cell carcinoma (RCC). The aim of this study was to examine the possible association of Epstein-Barr virus (EBV) infection with clinicopathological features and cellular biomarkers including p53, p16INK4a, Ki-67 and nuclear factor-kappa B (NF-κB) in RCC tumors. METHODS: In this prospective study, 122 histologically confirmed Formalin-fixed Paraffin-embedded RCC tissue specimens along with 96 specimens of their corresponding peritumoral tissues and 23 samples of blunt renal injuries were subjected to nested polymerase chain reaction (nPCR) in order to amplify EBV DNA sequences. The expression of p53, p16INK4a, Ki-67 and NF-κB was investigated by immunohistochemistry (IHC) assay. Statistical analysis was employed to demonstrate the possible associations. RESULTS: Infection with EBV was found to be significantly associated with RCC. Our results indicate that p65 NF-κB signaling pathway is probably involved in EBV-mediated RCC pathogenesis. Moreover, we found p53, Ki-67 and cytoplasmic NF-κB expression to be associated with tumor nuclear grade in RCC patients. The expression of p53 and Ki-67 was associated with primary tumor category as well. In addition, p53 overexpression was significantly more frequent among nonconventional RCC tumors than the conventional histologic type. CONCLUSIONS: Infection with EBV is likely to play an important role in the development of RCC through the constitutive and permanent activation of NF-κB p65 signaling pathway. However, more experiments and supporting data are required to reach a decisive conclusion.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Renais/virologia , Infecções por Vírus Epstein-Barr , Neoplasias Renais/virologia , NF-kappa B/análise , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Autoantígenos/análise , Autoantígenos/genética , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Criança , Pré-Escolar , Regulação Neoplásica da Expressão Gênica , Genes p53/genética , Humanos , Imuno-Histoquímica , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Pessoa de Meia-Idade , NF-kappa B/genética , Gradação de Tumores , Estudos Prospectivos , Complexo de Endopeptidases do Proteassoma/análise , Complexo de Endopeptidases do Proteassoma/genética , Transdução de Sinais , Adulto JovemRESUMO
Neuroblastoma is a neural crest-derived childhood tumor of the peripheral nervous system in which MycN amplification is a hallmark of poor prognosis. Here we show that MycN is expressed together with phosphorylation-stabilizing factor CIP2A in regions of the neural plate destined to form the CNS, but MycN is excluded from the neighboring neural crest stem cell domain. Interestingly, ectopic expression of MycN or CIP2A in the neural crest domain biases cells toward CNS-like neural stem cells that express Sox2. Consistent with this, some forms of neuroblastoma have been shown to share transcriptional resemblance with CNS neural stem cells. As high MycN/CIP2A levels correlate with poor prognosis, we posit that a MycN/CIP2A-mediated cell-fate bias may reflect a possible mechanism underlying early priming of some aggressive forms of neuroblastoma. In contrast to MycN, its paralogue cMyc is normally expressed in the neural crest stem cell domain and typically is associated with better overall survival in clinical neuroblastoma, perhaps reflecting a more "normal" neural crest-like state. These data suggest that priming for some forms of aggressive neuroblastoma may occur before neural crest emigration from the CNS and well before sympathoadrenal specification.
Assuntos
Autoantígenos/fisiologia , Proteínas de Membrana/fisiologia , Proteína Proto-Oncogênica N-Myc/fisiologia , Crista Neural/citologia , Células-Tronco Neurais/fisiologia , Neuroblastoma/etiologia , Autoantígenos/análise , Movimento Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/análise , Proteína Proto-Oncogênica N-Myc/análise , Neuroblastoma/patologia , Fatores de Transcrição SOXB1/análiseRESUMO
Membranous nephropathy is characterized by deposition of immune complexes along the glomerular basement membrane. PLA2R and THSD7A are target antigens in 70% and 1-5% of primary membranous nephropathy cases, respectively. In the remaining cases, the target antigen is unknown. Here, laser microdissection of glomeruli followed by mass spectrometry was used to identify novel antigen(s) in PLA2R-negative membranous nephropathy. An initial pilot mass spectrometry study in 35 cases of PLA2R-negative membranous nephropathy showed high spectral counts for neural tissue encoding protein with EGF-like repeats, NELL-1, in six cases. Mass spectrometry failed to detect NELL-1 in 23 PLA2R-associated membranous nephropathy and 88 controls. NELL-1 was localized by immunohistochemistry, which showed bright granular glomerular basement membrane staining for NELL-1 in all six cases. Next, an additional 23 NELL-1 positive cases of membranous nephropathy were identified by immunohistochemistry in a discovery cohort of 91 PLA2R-negative membranous nephropathy cases, 14 were confirmed by mass spectrometry. Thus, 29 of 126 PLA2R-negative cases were positive for NELL-1. PLA2R-associated membranous nephropathy and controls stained negative for NELL-1. We then identified five NELL-1 positive cases of membranous nephropathy out of 84 PLA2R and THSD7A-negative cases in two validation cohorts from France and Belgium. By confocal microscopy, both IgG and NELL-1 co-localized to the glomerular basement membrane. Western blot analysis showed reactivity to NELL-1 in five available sera, but no reactivity in control sera. Clinical and biopsy findings of NELL-1 positive membranous nephropathy showed features of primary membranous nephropathy. Thus, a subset of membranous nephropathy is associated with accumulation and co-localization of NELL-1 and IgG along the glomerular basement membrane, and with anti-NELL-1 antibodies in the serum. Hence, NELL-1 defines a distinct type of primary membranous nephropathy.
Assuntos
Autoantígenos/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Membrana Basal Glomerular/patologia , Glomerulonefrite Membranosa/imunologia , Idoso , Autoanticorpos/análise , Autoanticorpos/sangue , Autoanticorpos/imunologia , Autoantígenos/análise , Biópsia , Proteínas de Ligação ao Cálcio/análise , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Membrana Basal Glomerular/imunologia , Membrana Basal Glomerular/ultraestrutura , Glomerulonefrite Membranosa/sangue , Glomerulonefrite Membranosa/diagnóstico , Glomerulonefrite Membranosa/patologia , Humanos , Microdissecção e Captura a Laser , Masculino , Espectrometria de Massas , Microscopia Confocal , Microscopia Eletrônica , Microscopia de Fluorescência , Pessoa de Meia-Idade , Projetos Piloto , Receptores da Fosfolipase A2/análise , Receptores da Fosfolipase A2/imunologia , Trombospondinas/análise , Trombospondinas/imunologiaRESUMO
The ability to distinguish malignant from indolent prostate cancer cells is critically important for identification of clinically significant prostate cancer to minimize unnecessary overtreatment and sufferings endured by patients who have indolent cancer. Recently, we discovered that loss of giantin function as the primary Golgi targeting site for endoplasmic reticulum-derived transport vesicles in aggressive prostate cancer cells caused a shift of the Golgi localization site of α-mannosidase 1A to 130 KDa Golgi matrix protein (GM130)-65 KDa Golgi reassembly-stacking protein (GRASP65) site resulting in emergence of high mannose N-glycans on trans-Golgi enzymes and cell surface glycoproteins. To extend this observation, we isolated two cell clones (Clone 1 and Clone 2) from high passage LNCaP cells, which exhibited androgen refractory property missing in low passage LNCaP cells, and characterized their malignant property. We have found that comparing to Clone 2, which does not have cell surface high mannose N-glycans and exhibits localization of α-mannosidase 1A at giantin site, Clone 1 displays cell surface high mannose N-glycans, exhibits localization of α-mannosidase 1A at GM130-GRASP65 site, and shows a faster rate of closing the wound in a wound healing assay. The results indicate that Golgi localization of α-mannosidase 1A at GM130-GRASP65 site and appearance of cell surface high mannose N-glycans may serve as markers of malignant prostate cancer cells.
Assuntos
Autoantígenos/análise , Complexo de Golgi/patologia , Proteínas da Matriz do Complexo de Golgi/análise , Manose/análise , Proteínas de Membrana/análise , Neoplasias da Próstata/patologia , Biomarcadores Tumorais/análise , Linhagem Celular Tumoral , Membrana Celular/patologia , Humanos , Masculino , Polissacarídeos/análiseRESUMO
OBJECTIVE: Complement components, including C4d, can be found on activated platelets, a process associated with vascular disease in SLE. We investigated whether platelet C4d (PC4d) adds additional value to traditional and known lupus-associated risk factors when identifying SLE patients with vascular disease. METHODS: This cross-sectional study included 308 well-characterized SLE patients and 308 matched general population controls. PC4d deposition was analysed using flow cytometry. Values >95% of controls were considered as PC4d positive (+). aPL were determined by Luminex, and the LA test was performed by DRVVT. History of vascular disease (composite and as separate outcomes) was defined at inclusion. RESULTS: SLE patients had increased PC4d deposition as compared with population controls (50 vs 5%, P < 0.0001). PC4d+ positively associated with any vascular events, and separately with venous and cerebrovascular events, and also with all investigated aPL profiles. The association for any vascular event remained statistically significant after adjustment for traditional and SLE-associated risk factors (odds ratio: 2.3, 95% CI: 1.3, 4.3, P = 0.008). Compared with patients negative for both PC4d and LA, patients with double positivity were more likely to have vascular disease (odds ratio: 12.3, 95% CI: 5.4, 29.3; attributable proportion due to interaction 0.8, 95% CI: 0.4, 1.1). CONCLUSION: PC4d+ is associated with vascular events in SLE, independently of traditional and SLE-associated risk factors. Concurrent presence of PC4d and LA seem to interact to further increase the odds for vascular events. Prospective studies should examine whether the aPL/PC4d combination can improve prediction of vascular events in SLE and/or APS.
Assuntos
Plaquetas/imunologia , Complemento C4b/análise , Lúpus Eritematoso Sistêmico/imunologia , Fragmentos de Peptídeos/análise , Doenças Vasculares/imunologia , Adulto , Autoantígenos/análise , Biomarcadores/análise , Doenças das Artérias Carótidas/diagnóstico por imagem , Estudos de Casos e Controles , Ativação do Complemento , Estudos Transversais , Feminino , Citometria de Fluxo , Humanos , Lúpus Eritematoso Sistêmico/complicações , Masculino , Pessoa de Meia-Idade , Razão de Chances , Ribonucleoproteínas/análise , Fatores de Risco , Doenças Vasculares/etiologia , Antígeno SS-BRESUMO
Narcolepsy type 1 (NT1) is a chronic sleep disorder caused by a specific loss of hypocretin-producing neurons. The incidence of NT1 increased in Sweden, Finland and Norway following Pandemrix®-vaccination, initiated to prevent the 2009 influenza pandemic. The pathogenesis of NT1 is poorly understood, and causal links to vaccination are yet to be clarified. The strong association with Human leukocyte antigen (HLA) DQB1*06:02 suggests an autoimmune pathogenesis, but proposed autoantigens remain controversial. We used a two-step approach to identify autoantigens in patients that acquired NT1 after Pandemrix®-vaccination. Using arrays of more than 9000 full-length human proteins, we screened the sera of 10 patients and 24 healthy subjects for autoantibodies. Identified candidate antigens were expressed in vitro to enable validation studies with radiobinding assays (RBA). The validation cohort included NT1 patients (n = 39), their first-degree relatives (FDR) (n = 66), population controls (n = 188), and disease controls representing multiple sclerosis (n = 100) and FDR to type 1 diabetes patients (n = 41). Reactivity towards previously suggested NT1 autoantigen candidates including Tribbles homolog 2, Prostaglandin D2 receptor, Hypocretin receptor 2 and α-MSH/proopiomelanocortin was not replicated in the protein array screen. By comparing case to control signals, three novel candidate autoantigens were identified in the protein array screen; LOC401464, PARP3 and FAM63B. However, the RBA did not confirm elevated reactivity towards either of these proteins. In summary, three putative autoantigens in NT1 were identified by protein array screening. Autoantibodies against these candidates could not be verified with independent methods. Further studies are warranted to identify hypothetical autoantigens related to the pathogenesis of Pandemrix®-induced NT1.
Assuntos
Autoanticorpos/sangue , Autoantígenos/análise , Vacinas contra Influenza/efeitos adversos , Narcolepsia/imunologia , Análise Serial de Proteínas/métodos , Autoantígenos/imunologia , Feminino , Humanos , MasculinoRESUMO
Autism spectrum disorder (ASD) is an important health issue and affects 1 in 59 children in the US. Prior studies determined that maternal autoantibody related (MAR) autism is thought to be associated with ~23% of ASD cases. We previously identified seven MAR-specific autoantigens including CRMP1, CRMP2, GDA, LDHA, LDHB, STIP1, and YBX1. We subsequently described the epitope peptide sequences recognized by maternal autoantibodies for each of the seven ASD-specific autoantigens. The aim of the current study was to expand upon our previous work and identify additional antigens recognized by the ASD-specific maternal autoantibodies, as well as to map the unique ASD-specific epitopes using microarray technology. Fetal Rhesus macaque brain tissues were separated by molecular weight and a fraction containing bands between 37 and 45 kDa was analyzed using 2-D gel electrophoresis, followed by peptide mass mapping using MALDI-TOF MS and TOF/TOF tandem MS/MS. Using this methodology, Neuron specific enolase (NSE) was identified as a target autoantigen and selected for epitope mapping. The full NSE sequence was translated into 15-mer peptides with an overlap of 14 amino acids onto microarray slides and probed with maternal plasma from mothers with an ASD child and from mothers with a Typically Developing child (TD) (ASD = 27 and TD = 21). The resulting data were analyzed by T-test. We found 16 ASD-specific NSE-peptide sequences for which four sequences were statistically significant (p < 0.05) using both the t-test and SAM t-test: DVAASEFYRDGKYDL (p = 0.047; SAM score 1.49), IEDPFDQDDWAAWSK (p = 0.049; SAM score 1.49), ERLAKYNQLMRIEEE (p = 0.045; SAM score 1.57), and RLAKYNQLMRIEEEL (p = 0.017; SAM score 1.82). We further identified 5 sequences that were recognized by both ASD and TD antibodies suggesting a large immunodominant epitope (DYPVVSIEDPFDQDDWAAW). While maternal autoantibodies against the NSE protein are present both in mothers with ASD and mothers of TD children, there are several ASD-specific epitopes that can potentially be used as MAR ASD biomarkers. Further, studies including analysis of NSE as a target protein in combination with the previously identified MAR ASD autoantigens are currently underway.
Assuntos
Transtorno do Espectro Autista , Biomarcadores , Peptídeos , Fosfopiruvato Hidratase , Animais , Transtorno do Espectro Autista/sangue , Transtorno do Espectro Autista/diagnóstico , Autoanticorpos , Autoantígenos/análise , Biomarcadores/sangue , Criança , Mapeamento de Epitopos , Feminino , Humanos , Macaca mulatta , Peptídeos/análise , Fosfopiruvato Hidratase/sangue , Fosfopiruvato Hidratase/imunologia , Espectrometria de Massas em TandemRESUMO
The Mis18 complex has been identified as a critical factor for the centromeric localization of a histone H3 variant, centromeric protein A (CENP-A), which is responsible for the specification of centromere identity in the chromosome. However, the functional role of Mis18 complex is largely unknown. Here, we generated Mis18α conditional knockout mice and found that Mis18α deficiency resulted in lethality at early embryonic stage with severe defects in chromosome segregation caused by mislocalization of CENP-A. Further, we demonstrate Mis18α's crucial role for epigenetic regulation of centromeric chromatin by reinforcing centromeric localization of DNMT3A/3B. Mis18α interacts with DNMT3A/3B, and this interaction is critical for maintaining DNA methylation and hence regulating epigenetic states of centromeric chromatin. Mis18α deficiency led to reduced DNA methylation, altered histone modifications, and uncontrolled noncoding transcripts in centromere region by decreased DNMT3A/3B enrichment. Together, our findings uncover the functional mechanism of Mis18α and its pivotal role in mammalian cell cycle.
Assuntos
Autoantígenos/metabolismo , Centrômero/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/fisiologia , Segregação de Cromossomos/genética , Epigênese Genética , Animais , Autoantígenos/análise , Sítios de Ligação , Centrômero/metabolismo , Proteína Centromérica A , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/análise , Proteínas Cromossômicas não Histona/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA Metiltransferase 3A , Células HeLa , Histonas/metabolismo , Humanos , Camundongos , Camundongos Knockout , Mapeamento de Interação de ProteínasRESUMO
Autoantibodies related to central nervous system (CNS) diseases propel research on paraneoplastic neurological syndrome (PNS). This syndrome develops autoantibodies in combination with certain neurological syndromes and cancers, such as anti-HuD antibodies in encephalomyelitis with small cell lung cancer and anti-Yo antibodies in cerebellar degeneration with gynecological cancer. These autoantibodies have roles in the diagnosis of neurological diseases and early detection of cancers that are usually occult. Most of these autoantibodies have no pathogenic roles in neuronal dysfunction directly. Instead, antigen-specific cytotoxic T lymphocytes are thought to have direct roles in neuronal damage. The recent discoveries of autoantibodies against neuronal synaptic receptors/channels produced in patients with autoimmune encephalomyelitis have highlighted insights into our understanding of the variable neurological symptoms in this disease. It has also improved our understanding of intractable epilepsy, atypical psychosis, and some demyelinating diseases that are ameliorated with immune therapies. The production and motility of these antibodies through the blood-brain barrier into the CNS remains unknown. Most of these recently identified autoantibodies bind to neuronal and glial cell surface synaptic receptors, potentially altering the synaptic signaling process. The clinical features differ among pathologies based on antibody targets. The investigation of these antibodies provides a deeper understanding of the background of neurological symptoms in addition to novel insights into their basic neuroscience.
Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Encefalite/imunologia , Doença de Hashimoto/imunologia , Proteínas do Tecido Nervoso/imunologia , Antígenos de Superfície/imunologia , Autoanticorpos/análise , Autoantígenos/análise , Encefalite/patologia , Feminino , Doença de Hashimoto/patologia , Humanos , Masculino , Proteínas do Tecido Nervoso/análise , Doenças do Sistema Nervoso/imunologia , Doenças do Sistema Nervoso/patologia , Neuroglia/química , Neuroglia/imunologia , Neurônios/química , Neurônios/imunologia , Síndromes Paraneoplásicas do Sistema Nervoso/imunologia , Síndromes Paraneoplásicas do Sistema Nervoso/patologia , Receptores de Neurotransmissores/análise , Receptores de Neurotransmissores/imunologia , Frações Subcelulares/químicaRESUMO
We recently discovered hybrid insulin peptides (HIPs) as a novel class of post-translationally modified peptides in murine-derived beta cell tumors, and we demonstrated that these molecules are autoantigens in type 1 diabetes (T1D). A HIP consists of an insulin fragment linked to another secretory granule peptide via a peptide bond. We verified that autoreactive CD4 T cells in both mouse and human autoimmune diabetes recognize these modified peptides. Here, we use mass spectrometric analyses to confirm the presence of HIPs in both mouse and human pancreatic islets. We also present criteria for the confident identification of these peptides. This work supports the hypothesis that HIPs are autoantigens in human T1D and provides a foundation for future efforts to interrogate this previously unknown component of the beta cell proteome.
Assuntos
Autoantígenos/análise , Insulina/química , Ilhotas Pancreáticas/química , Espectrometria de Massas/métodos , Animais , Autoantígenos/sangue , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/metabolismo , Humanos , Camundongos , Peptídeos/análise , Peptídeos/químicaRESUMO
Germline-encoded receptors recognizing common pathogen-associated molecular patterns are a central element of the innate immune system and play an important role in shaping the host response to infection. Many of the innate immune molecules central to these signaling pathways are evolutionarily conserved. LysMD3 is a novel molecule containing a putative peptidoglycan-binding domain that has orthologs in humans, mice, zebrafish, flies, and worms. We found that the lysin motif (LysM) of LysMD3 is likely related to a previously described peptidoglycan-binding LysM found in bacteria. Mouse LysMD3 is a type II integral membrane protein that co-localizes with GM130+ structures, consistent with localization to the Golgi apparatus. We describe here two lines of mLysMD3-deficient mice for in vivo characterization of mLysMD3 function. We found that mLysMD3-deficient mice were born at Mendelian ratios and had no obvious pathological abnormalities. They also exhibited no obvious immune response deficiencies in a number of models of infection and inflammation. mLysMD3-deficient mice exhibited no signs of intestinal dysbiosis by 16S analysis or alterations in intestinal gene expression by RNA sequencing. We conclude that mLysMD3 contains a LysM with cytoplasmic orientation, but we were unable to define a physiological role for the molecule in vivo.
Assuntos
Deleção de Genes , Animais , Autoantígenos/análise , Infecções Bacterianas/genética , Infecções Bacterianas/imunologia , Sistemas CRISPR-Cas , Feminino , Imunidade Inata , Inflamação/genética , Inflamação/imunologia , Masculino , Proteínas de Membrana/análise , Camundongos , Micoses/genética , Micoses/imunologia , Filogenia , Viroses/genética , Viroses/imunologiaRESUMO
Sox13, a group D member of the Sry-related high-mobility group box (Sox) transcription factor family, is expressed in various tissues including the hair follicle. However, its spatiotemporal expression patterns in the hair follicle and its role in hair development remain to be elucidated. To address these questions, we generated Sox13-LacZ-knock-in mice (Sox13LacZ/+), in which the LacZ reporter gene was inserted in-frame into exon 2, which contains the translation initiation codon. X-gal staining in Sox13LacZ/+ embryos revealed that Sox13 is initially expressed in the epithelial portion of the placode, and subsequently in the hair germ and the hair peg during early hair follicle development. In postnatal catagen and anagen, Sox13 was detected in the epithelial sheath, whereas in telogen, Sox13 was localized in the bulge region, where hair follicle stem cells reside. Immunohistochemistry with an anti-ß-galactosidase antibody and anti-hair keratin antibodies that specifically mark the different layers of the hair follicle revealed that Sox13 was predominantly expressed in the outer root sheath in anagen. However, the integumentary structures of Sox13LacZ/LacZ mice were grossly and histologically indistinguishable from those of wild type mice. These results suggest that although Sox13 is dispensable for epidermal and adnexal development, Sox13 is a useful marker for early hair follicle development.
Assuntos
Autoantígenos/genética , Regulação da Expressão Gênica no Desenvolvimento , Folículo Piloso/crescimento & desenvolvimento , Análise Espaço-Temporal , Animais , Autoantígenos/análise , Biomarcadores , Conexinas , Embrião de Mamíferos , Folículo Piloso/embriologia , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Proteínas de Peixe-ZebraRESUMO
BACKGROUND: Mucous membrane pemphigoid (MMP) is a rare chronic autoimmune subepithelial blistering disorder, targeting multiple basement membrane zone (BMZ) proteins including collagen XVII (COL17). Circulating autoantibodies of MMP are often undetected due to their lower titers. The oral mucosa is a valuable substrate for the detection of autoantibodies in MMP patients. However, obtaining normal human oral mucosa is more difficult than obtaining normal human skin. We established immortalized normal human oral mucosal keratinocytes (OMKs) and performed immunoblotting using immortalized OMK lysate for detecting autoantigens in MMP. METHODS: Immortalized OMKs were generated from primary OMKs using E6/E7 proteins of HPV. We compared the protein expression levels of major BMZ proteins between primary OMKs and immortalized OMKs. We performed immunoblotting to detect autoantigens using cell lysates from immortalized OMKs in 30 MMP patients. RESULTS: There were no significant differences between primary OMKs and immortalized OMKs in terms of protein expression levels of the BMZ proteins, including COL17, laminin 332, integrin α6/ß4, collagen VII, and collagen IV. Cell lysates of immortalized OMKs effectively identified MMP autoantigens in 60% (18/30) of MMP sera. We found an interesting case of MMP whose autoantibodies preferentially reacted to the 120-kD protein that is an ectodomain of COL17. CONCLUSION: We demonstrated that a cell lysate of immortalized OMKs is a reliable substrate for the detection of MMP autoantigens. This newly developed immunoblotting analysis method promises to contribute to the diagnosis of MMP.
Assuntos
Autoantígenos/análise , Queratinócitos/imunologia , Mucosa Bucal/citologia , Penfigoide Mucomembranoso Benigno/diagnóstico , Penfigoide Mucomembranoso Benigno/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Autoantígenos/isolamento & purificação , Biomarcadores/análise , Feminino , Humanos , Immunoblotting/métodos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Atrial fibrillation (AF) is the most common chronic arrhythmia in elderly people and is accompanied by remodeling processes. While much is known about changes in ionic channels and in extracellular matrix, less is known about possible changes of intracellular structures. OBJECTIVE: We wanted to investigate, whether AF may also affect the structure of the Golgi apparatus and the microtubular network. METHODS: One-hundred fifty-three cardiac surgery patients were investigated [n = 24 in sinus rhythm (SR) and n = 129 with chronic AF of >1 year duration]. Tissue samples of the left atrial free wall were examined immunohistochemically. Golgi apparatus was detected by GM130 and its phosphorylated isoform. Furthermore, we investigated the length of the microtubules by α-tubulin staining. We also measured stathmin (phospho-S37), which is known to induce microtubule depolymerization. In addition, we investigated the cyclin-dependent kinase cdk5-activation, a typical stimulus for Golgi fragmentation, by measuring membrane-associated cdk5. RESULTS: We found significant fragmentation of the Golgi apparatus in AF together with a reduced fragment size. Significant more fragments of the Golgi were found lateral to the nucleus in AF, while the Golgi in SR was located more to the polar side of the nucleus, that is, in the longitudinal axis of the cell. This was accompanied by a significant reduction of the number of tubulin strands longer than 10 µm. These changes did not go along with an activation of stathmin, but with an increase in membrane association of cdk5. CONCLUSIONS: The present data may show that AF associated remodeling also involves intracellular remodeling of the Golgi-microtubular apparatus.
Assuntos
Fibrilação Atrial/patologia , Remodelamento Atrial , Complexo de Golgi/patologia , Átrios do Coração/patologia , Idoso , Fibrilação Atrial/metabolismo , Fibrilação Atrial/fisiopatologia , Autoantígenos/análise , Biomarcadores/análise , Estudos de Casos e Controles , Doença Crônica , Quinase 5 Dependente de Ciclina/análise , Feminino , Átrios do Coração/química , Átrios do Coração/fisiopatologia , Humanos , Masculino , Proteínas de Membrana/análise , Microtúbulos/química , Microtúbulos/patologia , Pessoa de Meia-Idade , Fosforilação , Estatmina/análise , Tubulina (Proteína)/análiseRESUMO
The dermal-epidermal junction (DEJ) provides a physical and biological interface between the epidermis and the dermis. In addition to providing a structural integrity, the DEJ also acts as a passageway for molecular transport. Based on the recently reported importance of the DEJ in skin aging, novel peptide derivatives have been tested for their effects on basement membrane (BM) protein expressions in cultured human epidermal keratinocytes. As a result, protein expressions of collagen XVII, laminin and nidogen were stimulated by the test peptide and peptides complex. Further ex vivo evaluation using excised human skin, confirmed that the topical application of the peptides complex significantly increased dermal collagen expression, as well as expressions of collagen XVII and laminin. Interestingly, while the origin of the laminin protein is epidermal keratinocytes, the immunohistochemical staining of skin showed that laminin was only detected in the uppermost layer of the dermis, which suggests a tight assembly of laminin protein onto the dermal side of the DEJ. These results suggest that a peptide complex could improve the structural properties of the DEJ through its ability to stimulate BM proteins. In order to evaluate the anti-wrinkle benefits of the peptide complex in vivo, a clinical study was performed on 22 healthy Asian female volunteers older than 40 years. As a result, significant improvements in skin wrinkles for all of the five sites were observed after two weeks, as assessed by skin topographic measurements. Collectively, these results demonstrate the anti-aging efficacy of the peptides complex.
Assuntos
Membrana Basal/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Peptídeos/farmacologia , Envelhecimento da Pele/efeitos dos fármacos , Pele/efeitos dos fármacos , Adulto , Autoantígenos/análise , Linhagem Celular , Colágeno Tipo I/análise , Feminino , Humanos , Queratinócitos/química , Queratinócitos/citologia , Laminina/análise , Pessoa de Meia-Idade , Colágenos não Fibrilares/análise , Pele/química , Pele/citologia , Colágeno Tipo XVIIRESUMO
Thickening of the glomerular basement membrane (GBM) and expansion of the mesangial matrix are hallmarks of diabetic nephropathy (DN), generally considered to emerge from different sites of overproduction: GBM components from podocytes and mesangial matrix from mesangial cells. Reevaluation of 918 biopsies with DN revealed strong evidence that these mechanisms are connected to each other, wherein excess GBM components fail to undergo degradation and are deposited in the mesangium. These data do not exclude that mesangial cells also synthesize components that contribute to the accumulation of matrix in the mesangium. Light, electron microscopic, immunofluorescence, and in situ hybridization studies clearly show that the thickening of the GBM is due not only to overproduction of components of the mature GBM (α3 and α5 chains of collagen IV and agrin) by podocytes but also to resumed increased synthesis of the α1 chain of collagen IV and of perlecan by endothelial cells usually seen during embryonic development. We hypothesize that these abnormal production mechanisms are caused by different processes: overproduction of mature GBM-components by the diabetic milieu and regression of endothelial cells to an embryonic production mode by decreased availability of mediators from podocytes.
Assuntos
Nefropatias Diabéticas/patologia , Membrana Basal Glomerular/ultraestrutura , Mesângio Glomerular/ultraestrutura , Podócitos/ultraestrutura , Agrina/análise , Autoantígenos/análise , Biópsia , Microambiente Celular , Colágeno Tipo IV/análise , Nefropatias Diabéticas/metabolismo , Progressão da Doença , Membrana Basal Glomerular/química , Mesângio Glomerular/química , Proteoglicanas de Heparan Sulfato/análise , Humanos , Imuno-Histoquímica , Microscopia Eletrônica de Transmissão , Podócitos/química , EscleroseRESUMO
During acute graft-versus-host disease (aGVHD) in mice, autoreactive T cells can be generated de novo in the host thymus implying an impairment in self-tolerance induction. As a possible mechanism, we have previously reported that mature medullary thymic epithelial cells (mTEC(high)) expressing the autoimmune regulator are targets of donor T-cell alloimmunity during aGVHD. A decline in mTEC(high) cell pool size, which purges individual tissue-restricted peripheral self-antigens (TRA) from the total thymic ectopic TRA repertoire, weakens the platform for central tolerance induction. Here we provide evidence in a transgenic mouse system using ovalbumin (OVA) as a model surrogate TRA that the de novo production of OVA-specific CD4(+) T cells during acute GVHD is a direct consequence of impaired thymic ectopic OVA expression in mTEC(high) cells. Our data, therefore, indicate that a functional compromise of the medullary mTEC(high) compartment may link alloimmunity to the development of autoimmunity during chronic GVHD.
Assuntos
Autoimunidade , Linfócitos T CD4-Positivos/patologia , Doença Enxerto-Hospedeiro/patologia , Tolerância a Antígenos Próprios , Timo/patologia , Animais , Autoantígenos/análise , Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Feminino , Doença Enxerto-Hospedeiro/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/imunologia , Timo/imunologiaRESUMO
Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A), a recently identified oncogene, has emerged as a potential drug target for a range of different tumor types. High CIP2A expression has been reported in almost all solid organ cancers and in some hematological tumors and is associated with high grade and poor prognosis. Notably, high CIP2A expression is determined in over 70% of tumor patient samples in the majority of human cancers. High expression of CIP2A has also been proposed as a useful biomarker that predicts therapeutic response to chemotherapeutics such as Bortezomib, Erlotinib, Checkpoint Kinase 1 inhibitors and pro-senescence based therapies. In this review, we highlight, critically evaluate and discuss the ambiguity in CIP2A's prognostic role in different human cancers and its role in modulating response and resistance to chemotherapeutics.
Assuntos
Autoantígenos/análise , Biomarcadores Tumorais/análise , Proteínas de Membrana/análise , Autoantígenos/fisiologia , Quinase 1 do Ponto de Checagem , Genes myc , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/fisiologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Prognóstico , Proteínas Quinases/fisiologiaRESUMO
The study of protein-protein interactions is an essential process to understand the biological functions of proteins and the underlying mechanisms. Co-immunoprecipitation coupled with mass spectrometry (CoIP-MS) is one of the most extensively used high-throughput techniques to discover novel protein-protein interactions. However, the traditional CoIP process uses whole cell lysate, disrupts cellular organization, and leads to potential false positives by inducing artificial protein-protein interactions. Here, we have developed a strategy by combining subcellular fractionation with CoIP-MS to study the interacting proteins of the complement component 1, q subcomponent binding protein (C1QBP) in the mitochondria. Using this method, a novel C1QBP interacting protein, dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial (DLAT) was identified and validated. Furthermore, the activity of the pyruvate dehydrogenase (PDH) was found to be affected by the expression level of C1QBP. These results provide novel insights regarding the mitochondrial function of C1QBP in the regulation of cellular energy metabolism. This method could also be used to analyze the subcellular protein-protein interactions for other proteins of interest.