RESUMO
INTRODUCTION: Tumor-associated status epilepticus (TASE) follows a relatively benign course compared with SE in the general population. Little, however, is known about associated prognostic factors. METHODS: We conducted a prospective, observational study of all cases of TASE treated at a tertiary hospital in Barcelona, Spain between May 2011 and May 2019. We collected data on tumor and SE characteristics and baseline functional status and analyzed associations with outcomes at discharge and 1-year follow-up. RESULTS: Eighty-two patients were studied; 58.5% (nâ¯=â¯48) had an aggressive tumor (glioblastoma or brain metastasis). Fifty-one patients (62.2%) had a favorable outcome at discharge compared with just 30 patients (25.8%) at 1-year follow-up. Fourteen patients (17.1%) died during hospitalization. Lateralized period discharges (LPDs) on the baseline electroencephalography (EEG), presence of metastasis, and SE severity were significantly associated with a worse outcome at discharge. The independent predictors of poor prognosis at 1-year follow-up were SE duration of at least 21â¯h, an aggressive brain tumor, and a nonsurgical treatment before SE onset. Lateralized period discharges, super-refractory SE, and an aggressive tumor type were independently associated with increased mortality. CONCLUSIONS: Status epilepticus duration is the main modifiable factor associated with poor prognosis at 1-year follow-up. Accordingly, patients with TASE, like those with SE of any etiology, should receive early, aggressive treatment.
Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/mortalidade , Hospitalização/tendências , Estado Epiléptico/diagnóstico por imagem , Estado Epiléptico/mortalidade , Centros de Atenção Terciária/tendências , Adulto , Idoso , Neoplasias Encefálicas/fisiopatologia , Estudos de Coortes , Eletroencefalografia/tendências , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Espanha/epidemiologia , Estado Epiléptico/fisiopatologia , Taxa de Sobrevida/tendênciasRESUMO
Although imaging the live Trypanosoma cruzi parasite is a routine technique in most laboratories, identification of the parasite in infected tissues and organs has been hindered by their intrinsic opaque nature. We describe a simple method for in vivo observation of live single-cell Trypanosoma cruzi parasites inside mammalian host tissues. BALB/c or C57BL/6 mice infected with DsRed-CL or GFP-G trypomastigotes had their organs removed and sectioned with surgical blades. Ex vivo organ sections were observed under confocal microscopy. For the first time, this procedure enabled imaging of individual amastigotes, intermediate forms and motile trypomastigotes within infected tissues of mammalian hosts.
Assuntos
Análise de Célula Única/métodos , Trypanosoma cruzi/citologia , Trypanosoma cruzi/patogenicidade , Animais , Doença de Chagas/parasitologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia ConfocalRESUMO
Microorganisms use specialized systems to export virulence factors into host cells. Secretion of effector proteins into the extracellular environment has been described in Trypanosoma cruzi; however, a comprehensive proteomic analysis of the secretome and the secretion mechanisms involved remain elusive. Here, we present evidence that T. cruzi releases proteins associated with vesicles that are formed by at least two different mechanisms. Transmission electron microscopy showed larger vesicles budding from the plasma membrane of noninfective epimastigotes and infective metacyclic trypomastigotes, as well as smaller vesicles within the flagellar pocket of both forms. Parasite conditioned culture supernatant was fractionated and characterized by morphological, immunochemical, and proteomic analyses. Three fractions were obtained by differential ultracentrifugation: the first enriched in larger vesicles resembling ectosomes, the second enriched in smaller vesicles resembling exosomes, and a third fraction enriched in soluble proteins not associated with extracellular vesicles. Label-free quantitative proteomic analysis revealed a rich collection of proteins involved in metabolism, signaling, nucleic acid binding, and parasite survival and virulence. These findings support the notion that T. cruzi uses different secretion pathways to excrete/secrete proteins. Moreover, our results suggest that metacyclic forms may use extracellular vesicles to deliver cargo into host cells.
Assuntos
Membrana Celular/metabolismo , Flagelos/metabolismo , Estágios do Ciclo de Vida/fisiologia , Proteínas de Protozoários/isolamento & purificação , Trypanosoma cruzi/fisiologia , Fatores de Virulência/isolamento & purificação , Animais , Transporte Biológico , Células Cultivadas , Cromatografia Líquida , Meios de Cultivo Condicionados/química , Camundongos , Microscopia Eletrônica de Transmissão , Proteômica , Proteínas de Protozoários/metabolismo , Vesículas Secretórias , Espectrometria de Massas em Tandem , UltracentrifugaçãoRESUMO
T. cruzi improves the likelihood of invading or adapting to the host through its capacity to present a large repertoire of surface molecules. The metacyclic stage-specific surface glycoprotein GP82 has been implicated in host cell invasion. GP82 is encoded by multiple genes from the trans-sialidase superfamily. GP82 shows a modular organization, with some variation of N-terminal region flanking a conserved central core where the binding sites to the mammalian cell and gastric mucin are located. The function of GP82 as adhesin in host cell invasion process could expose the protein to an intense conservative and selective pressure. GP82 is a GPI-anchored surface protein, synthesized as a 70 kDa precursor devoid of N-linked sugars. GPI-minus variants accumulate in the ER indicating that GPI anchor acts as a forward transport signal for progressing along the secretory pathway as suggested for T. cruzi mucins. It has been demonstrated that the expression of GP82 is constitutive and may be regulated at post-transcriptional level, for instance, at translational level and/or mRNA stabilization. GP82 mRNAs are mobilized to polysomes and consequently translated, but only in metacyclic trypomastigotes. Analysis of transgenic parasites indicates that the mechanism regulating GP82 expression involves multiple elements in the 3'UTR.
Assuntos
Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/fisiologia , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Sítios de Ligação , Adesão Celular , Doença de Chagas/parasitologia , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Família Multigênica , Polirribossomos/metabolismo , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidadeRESUMO
Introduction: Different medical therapies have been developed for pituitary adenomas. However, Non-Functioning Pituitary Neuroendocrine Tumors (NF-PitNET) have shown little response to them. Furthermore, epithelial-mesenchymal transition (EMT) has been linked to resistance to medical treatment in a significant number of tumors, including pituitary adenomas. Methods: We aimed to evaluate the expression of EMT-related markers in 72 NF-PitNET and 16 non-tumoral pituitaries. To further explore the potential usefulness of medical treatment for NF-PitNET we assessed the expression of somatostatin receptors and dopamine-associated genes. Results: We found that SNAI1, SNAI2, Vimentin, KLK10, PEBP1, Ki-67 and SSTR2 were associated with invasive NF-PitNET. Furthermore, we found that the EMT phenomenon was more common in NF-PitNET than in GH-secreting pituitary tumors. Interestingly, PEBP1 was overexpressed in recurrent NF-PitNET, and could predict growth recurrence with 100% sensitivity but only 43% specificity. In parallel with previously reported studies, SSTR3 is highly expressed in our NF-PitNET cohort. However, SSTR3 expression is highly heterogeneous among the different histological variants of NF-PitNET with very low levels in silent corticotroph adenomas. Conclusion: NF-PitNET showed an enhanced EMT phenomenon. SSTR3 targeting could be a good therapeutic candidate in NF-PitNET except for silent corticotroph adenomas, which express very low levels of this receptor. In addition, PEBP1 could be an informative biomarker of tumor regrowth, useful for predictive medicine in NF-PitNET.
Assuntos
Adenoma Hipofisário Secretor de ACT , Adenoma , Tumores Neuroendócrinos , Neoplasias Hipofisárias , Humanos , Neoplasias Hipofisárias/tratamento farmacológico , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/metabolismo , Tumores Neuroendócrinos/tratamento farmacológico , Tumores Neuroendócrinos/genética , Transição Epitelial-Mesenquimal/genética , Adenoma/tratamento farmacológico , Adenoma/genética , Adenoma/metabolismoRESUMO
The therapeutic benefit of approved BRAF and MEK inhibitors (BRAFi/MEKi) in patients with brain metastatic BRAF V600E/K-mutated melanoma is limited and transient. Resistance largely occurs through the restoration of MAPK signaling via paradoxical BRAF activation, highlighting the need for more effective therapeutic options. Aiming to address this clinical challenge, we characterized the activity of a potent, brain-penetrant paradox breaker BRAFi (compound 1a, C1a) as first-line therapy and following progression upon treatment with approved BRAFi and BRAFi/MEKi therapies. C1a activity was evaluated in vitro and in vivo in melanoma cell lines and patient-derived models of BRAF V600E-mutant melanoma brain metastases following relapse after treatment with BRAFi/MEKi. C1a showed superior efficacy compared with approved BRAFi in both subcutaneous and brain metastatic models. Importantly, C1a manifested potent and prolonged antitumor activity even in models that progressed on BRAFi/MEKi treatment. Analysis of mechanisms of resistance to C1a revealed MAPK reactivation under drug treatment as the predominant resistance-driving event in both subcutaneous and intracranial tumors. Specifically, BRAF kinase domain duplication was identified as a frequently occurring driver of resistance to C1a. Combination therapies of C1a and anti-PD-1 antibody proved to significantly reduce disease recurrence. Collectively, these preclinical studies validate the outstanding antitumor activity of C1a in brain metastasis, support clinical investigation of this agent in patients pretreated with BRAFi/MEKi, unveil genetic drivers of tumor escape from C1a, and identify a combinatorial treatment that achieves long-lasting responses. SIGNIFICANCE: A brain-penetrant BRAF inhibitor demonstrates potent activity in brain metastatic melanoma, even upon relapse following standard BRAF inhibitor therapy, supporting further investigation into its clinical utility.
Assuntos
Neoplasias Encefálicas , Melanoma , Encéfalo/patologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno , Mutação , Recidiva Local de Neoplasia/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-rafRESUMO
T-cell bispecific antibodies (TCB) are engineered molecules that bind both the T-cell receptor and tumor-specific antigens. Epidermal growth factor receptor variant III (EGFRvIII) mutation is a common event in glioblastoma (GBM) and is characterized by the deletion of exons 2-7, resulting in a constitutively active receptor that promotes cell proliferation, angiogenesis, and invasion. EGFRvIII is expressed on the surface of tumor cells and is not expressed in normal tissues, making EGFRvIII an ideal neoantigen target for TCBs. We designed and developed a novel 2+1 EGFRvIII-TCB with optimal pharmacologic characteristics and potent antitumor activity. EGFRvIII-TCB showed specificity for EGFRvIII and promoted tumor cell killing as well as T-cell activation and cytokine secretion only in patient-derived models expressing EGFRvIII. Moreover, EGFRvIII-TCB promoted T-cell recruitment into intracranial tumors. EGFRvIII-TCB induced tumor regression in GBM animal models, including humanized orthotopic GBM patient-derived xenograft models. Our results warrant the clinical testing of EGFRvIII-TCB for the treatment of EGFRvIII-expressing GBMs.
Assuntos
Anticorpos Biespecíficos , Neoplasias Encefálicas , Glioblastoma , Animais , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Citocinas , Receptores ErbB/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/metabolismoRESUMO
Deregulation of miRNAs contributes to the development of distinct cancer types, including melanoma, an aggressive form of skin cancer characterized by high metastatic potential and poor prognosis. The expression of a set of 580 miRNAs was investigated in a model of murine melanoma progression, comprising non-metastatic (4C11-) and metastatic melanoma (4C11+) cells. A significant increase in miR-138-5p expression was found in the metastatic 4C11+ melanoma cells compared to 4C11-, which prompted us to investigate its role in melanoma aggressiveness. Functional assays, including anoikis resistance, colony formation, collective migration, serum-deprived growth capacity, as well as in vivo tumor growth and experimental metastasis were performed in 4C11- cells stably overexpressing miR-138-5p. miR-138-5p induced an aggressive phenotype in mouse melanoma cell lines leading to increased proliferation, migration and cell viability under stress conditions. Moreover, by overexpressing miR-138-5p, low-growing and non-metastatic 4C11- cells became highly proliferative and metastatic in vivo, similar to the metastatic 4C11+ cells. Luciferase reporter analysis identified the tumor suppressor Trp53 as a direct target of miR-138-5p. Using data sets from independent melanoma cohorts, miR-138-5p and P53 expression were also found deregulated in human melanoma samples, with their levels negatively and positively correlated with prognosis, respectively. Our data shows that the overexpression of miR-138-5p contributes to melanoma metastasis through the direct suppression of Trp53.
Assuntos
Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Melanoma/mortalidade , MicroRNAs/genética , Interferência de RNA , Proteína Supressora de Tumor p53/genética , Regiões 3' não Traduzidas , Animais , Linhagem Celular Tumoral , Humanos , Melanoma/patologia , Camundongos , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Análise de SobrevidaRESUMO
Brain metastases are the most common tumor of the brain with a dismal prognosis. A fraction of patients with brain metastasis benefit from treatment with immune checkpoint inhibitors (ICI) and the degree and phenotype of the immune cell infiltration has been used to predict response to ICI. However, the anatomical location of brain lesions limits access to tumor material to characterize the immune phenotype. Here, we characterize immune cells present in brain lesions and matched cerebrospinal fluid (CSF) using single-cell RNA sequencing combined with T cell receptor genotyping. Tumor immune infiltration and specifically CD8+ T cell infiltration can be discerned through the analysis of the CSF. Consistently, identical T cell receptor clonotypes are detected in brain lesions and CSF, confirming cell exchange between these compartments. The analysis of immune cells of the CSF can provide a non-invasive alternative to predict the response to ICI, as well as identify the T cell receptor clonotypes present in brain metastasis.
Assuntos
Neoplasias Encefálicas/imunologia , Líquido Cefalorraquidiano/imunologia , Leucócitos , Microambiente Tumoral/imunologia , Adenocarcinoma de Pulmão , Encéfalo/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linfócitos T CD8-Positivos/imunologia , Humanos , Inibidores de Checkpoint Imunológico , Neoplasias Pulmonares , PrognósticoRESUMO
INTRODUCTION: The aim of this article was to evaluate the effectiveness of the Gleason grade groups (GGG) system on a group of Argentinian patients with prostate cancer (PC) who underwent radical prostatectomy (RP). MATERIAL AND METHODS: We retrospectively studied 262 patients who underwent RP between 1996 and 2014. To determine the performance and validity of the GGG system, a Kaplan-Meier analysis and multivariate analysis with Cox proportional method were performed to evaluate biochemical recurrence, distance metastases and specific cancer mortality. The area under the curve (AUC) was calculated to compare new groups of degrees of the GGG system with the classical scheme of stratification into 3 groups. RESULTS: The median follow-up was 84 months. As the groups ascend, there is less confined organ disease (p <0.001) and greater extraprostatic extension (p <0.001), greater invasion of seminal vesicles (p <0.001) and greater lymph node involvement (p <0.001). The biochemical recurrence-free survival at 5 years was 68%, 55%, 22%, 9%, 0% of the 1-5 groups, respectively. Ten-years cancer-specific survival was 96%, 95%, 78%, 64%, 25% for group 1-5, respectively. In the multivariate analysis, the GGG system is presented as the only independent predictor of biochemical recurrence and specific cancer mortality. The AUC indicates that the GGG system has a higher prognostic discrimination compared to the classic 3-group system (6, 7, ≥8). CONCLUSIONS: The International Society of Urological Pathology (ISUP) GGG system is an independent predictor of biochemical recurrence and mortality from prostate cancer in patients treated with RP. The classification into 5 groups shows greater discrimination in the prognosis than the traditional Gleason classification.
RESUMO
The adverse environmental conditions found in the periodontium during periodontitis pathogenesis stimulate local autophagy responses, mainly due to a continuous inflammatory response against the dysbiotic subgingival microbiome. The junctional epithelium represents the main site of the initial interaction between the host and the dysbiotic biofilm. Here, we investigated the role of autophagy in junctional epithelium keratinocytes (JEKs) in response to Aggregatibacter actinomycetemcomitans or its purified lipopolysaccharides (LPS). Immunofluorescence confocal analysis revealed an extensive nuclear translocation of transcription factor EB (TFEB) and consequently, an increase in autophagy markers and LC3-turnover assessed by immunoblotting and qRT-PCR. Correspondingly, challenged JEKs showed a punctuate cytosolic profile of LC3 protein contrasting with the diffuse distribution observed in untreated controls. Three-dimensional reconstructions of confocal images displayed a close association between intracellular bacteria and LC3-positive vesicles. Similarly, a close association between autophagic vesicles and the protein p62 was observed in challenged JEKs, indicating that p62 is the main adapter protein recruited during A. actinomycetemcomitans infection. Finally, the pharmacological inhibition of autophagy significantly increased the number of bacteria-infected cells as well as their death, similar to treatment with LPS. Our results indicate that A. actinomycetemcomitans infection induces autophagy in JEKs, and this homeostatic process has a cytoprotective effect on the host cells during the early stages of infection.
Assuntos
Aggregatibacter actinomycetemcomitans/fisiologia , Autofagia , Inserção Epitelial/patologia , Queratinócitos/microbiologia , Queratinócitos/patologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Biomarcadores/metabolismo , Contagem de Células , Linhagem Celular , Núcleo Celular/metabolismo , Sobrevivência Celular , Humanos , Imageamento Tridimensional , Lipopolissacarídeos/isolamento & purificação , Modelos Biológicos , Transporte Proteico , Proteína Sequestossoma-1/metabolismoRESUMO
BACKGROUND: Myxopapillary ependimoma (MPE) is a benign slow-growing tumor, and it has been designated histologically as a Grade I neoplasm according to the 2016 World Health Organization classification. Despite the benign character, dissemination and metastasis have occasionally been reported. The retrograde dissemination to other levels of the neuraxis is extremely rare, being more frequent to the intracranial compartment. CASE DESCRIPTION: We hereby present a case of medullary metastasis of cauda equina MPE, with a history of having undergone a subtotal resection and postoperative adjuvant radiotherapy. The patient presents complaints of night dorsal pain attributable to intradural metastasis twenty-one years after the first surgical intervention. CONCLUSION: The case reported highlights the importance of long follow-up in patients with MPE, since the possibility of secondary seeding to distant craniospinal sites or local spinal sites after surgery, and radiotherapy should be considered in metastatic disease.
RESUMO
Trypanosoma cruzi, the causative agent of Chagas disease, has a dense coat of GPI-anchored virulence factors. T. cruzi GPI-anchored adhesin GP82 is encoded by a repertoire of transcripts containing several in-frame initiation codons located up-stream from that adjacent to the predicted signal peptide (SP). Transfection of T. cruzi epimastigotes with constructs encoding GP82 starting at the SP or from the farthest up-stream methionine confirmed protein expression on the parasite cell surface, comparable to the native GP82. Proteins were fully functional, inducing parasite adhesion to HeLa cells and lysosome mobilization, events required for parasite invasion. Transgenic and native GP82 proteins showed indistinguishable electrophoretic mobility, suggesting similar processing of the SP. Deletion of SP generated a ~72 kDa protein devoid of N-linked oligosaccharides allowing irrefutable identification of GP82 precursor. SP transposition to an internal region of GP82 rendered the signal unrecognizable by the signal peptidase and incapable to direct the nascent protein for ER-membrane association. Altogether our data strongly suggests that GP82 SP fails to function as transmembrane domain and its recognition by the signal peptidase shows strict dependence on the signal localization at protein N-terminus. This report presents the first experimental characterization of the full-length GP82 and its signal peptide.
Assuntos
Doença de Chagas/patologia , Sinais Direcionadores de Proteínas/genética , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/patogenicidade , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo , Fatores de Virulência/metabolismo , Doença de Chagas/parasitologia , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Proteínas de Protozoários/genética , Alinhamento de Sequência , Relação Estrutura-Atividade , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Fatores de Virulência/genéticaRESUMO
Las enfermedades causadas por amebas de vida libre son infecciones oportunistas que pueden tener un curso fatal. Pueden producir afecciones diseminadas graves con compromiso del sistema nervioso central, como la encefalitis amebiana granulomatosa. Esta infección es cada vez más frecuente en América Latina, aunque se reconocen tardíamente debido a la similitud con otras patologías o porque es inusual incluirla en el diagnóstico diferencial. Comunicamos un caso fatal de una encefalitis amebiana granulomatosa por Balamuthia mandrillaris en una niña de 10 años. Destacamos la gravedad de la afectación cerebral y la falta de esquemas antimicrobianos validados para su tratamiento. Hoy en el mundo esta infección es considerada una enfermedad emergente, influenciada por el cambio climático, lo que llama a estar atentos a su presencia.
Diseases caused by free-living amoebae are opportunistic infections that can have a fatal course. They can cause very serious disseminated conditions with involvement of the central nervous system such as granulomatous amoebic encephalitis. This infection has become more common in Latin America, although its recognition is late due to the similarity with other pathological conditions or because it is unusual to include it in the differential diagnosis. We report a fatal case of granulomatous amoebic encephalitis due to Balamuthia mandrillaris in a 10-year-old girl. We highlight the severity of the brain involvement and the lack of validated schemes for its treatment. Today in the world this infection is considered an emerging disease, influenced by climate change, which calls for being attentive to its presence.
Assuntos
Humanos , Feminino , Criança , Encefalite Infecciosa/diagnóstico , Amebíase/diagnóstico , Tomografia Computadorizada por Raios X , Análise de Sequência de DNA , Evolução Fatal , Balamuthia mandrillaris/isolamento & purificação , Balamuthia mandrillaris/genética , Encefalite Infecciosa/diagnóstico por imagem , Amebíase/diagnóstico por imagemRESUMO
During Trypanosoma cruzi cell invasion, signal transduction pathways are triggered in parasite and host cells, leading to a rise in intracellular Ca2+ concentration. We posed the question whether calcineurin (CaN), in particular the functional regulatory subunit CaNB, a Ca2+-binding EF-hand protein, was expressed in T. cruzi and whether it played a role in cell invasion. Here we report the cloning and characterization of CL strain CaNB gene, as well as the participation of CaNB in cell invasion. Treatment of metacyclic trypomastigotes (MT) or tissue-culture trypomastigotes (TCT) with the CaN inhibitors cyclosporin or cypermethrin strongly inhibited (62-64%) their entry into HeLa cells. In assays using anti-phospho-serine/threonine antibodies, a few proteins of MT were found to be dephosphorylated in a manner inhibitable by cyclosporin upon exposure to HeLa cell extract. The phosphatase activity of CaN was detected by a biochemical approach in both MT and TCT. Treatment of parasites with antisense phosphorothioate oligonucleotides directed to TcCaNB-CL, which reduced the expression of TcCaNB and affected TcCaN activity, resulted in approximately 50% inhibition of HeLa cell entry by MT or TCT. Given that TcCaNB-CL may play a key role in cell invasion and differs considerably in its primary structure from the human CaNB, it might be considered as a potential chemotherapeutic target.
Assuntos
Calcineurina/fisiologia , Proteínas de Protozoários/fisiologia , Trypanosoma cruzi/patogenicidade , Fatores de Virulência/fisiologia , Sequência de Aminoácidos , Animais , Calcineurina/biossíntese , Calcineurina/genética , Inibidores de Calcineurina , Clonagem Molecular , Ciclosporina/farmacologia , Inibidores Enzimáticos/farmacologia , Inativação Gênica , Células HeLa , Humanos , Dados de Sequência Molecular , Oligonucleotídeos Antissenso , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/biossíntese , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/fisiologia , Filogenia , Proteínas de Protozoários/biossíntese , Piretrinas , Alinhamento de Sequência , Fatores de Virulência/biossínteseRESUMO
Trypanosoma cruzi, the parasite causing Chagas' disease, relies on triatomines for its transmission. T. cruzi metacyclic trypomastigotes express GP82 and GP90, which are developmentally regulated surface proteins that have been implicated in host cell invasion. We used quantitative RT-PCR to quantify GP90 and GP82 mRNA levels expressed by T. cruzi in the digestive tract of experimentally infected Rhodnius prolixus at different times post infection. Translation of these transcripts was assessed by immunofluorescence using specific monoclonal antibodies against GP90 and GP82. We found that although GP82 and GP90 proteins were not detected in epimastigote cells by immunofluorescence, transcripts were present at lower levels. Increased levels of GP90 and GP82 transcripts and the appearance of these proteins on the parasite surface were accompanied by morphological differentiation from epimastigotes into metacyclic forms. Our data suggest that during in vivo metacyclogenesis there is a coordinated mechanism that links stabilization of GP90 and GP82 mRNAs with their translation.
Assuntos
Trato Gastrointestinal/parasitologia , Insetos Vetores/parasitologia , Glicoproteínas de Membrana/biossíntese , Proteínas de Protozoários/biossíntese , Rhodnius/parasitologia , Trypanosoma cruzi/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/biossíntese , Animais , Expressão Gênica , Glicoproteínas de Membrana/genética , Microscopia de Fluorescência , Proteínas de Protozoários/genética , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glicoproteínas Variantes de Superfície de Trypanosoma/genéticaRESUMO
INTRODUCTION: The aim of this study was to describe the prognostic impact of microvascular invasion (MVI) in patients with non-metastatic renal cell cancer. MATERIAL AND METHODS: We carried out a retrospective, descriptive and analytical study of patients with non-metastatic renal cell carcinoma who had undergone a radical or partial nephrectomy. Patients were divided according to the presence of MVI. In each group, clinical and pathological characteristics were evaluated. Metastasis-free and cancer-specific survival was evaluated by the Kaplan Meier method. The multivariate analysis was performed with Cox proportional method in order to predict risk factors of metastasis and cancer-specific mortality. RESULTS: A total of 221 patients with a median of 40-month long follow-up were evaluated. Patients with MVI+ were 40 (18%) while those with MVI - were 181 (82%). In the univariate analysis, the presence of MVI had a strong correlation with symptomatic tumors (OR 3.56; p 0.0003), tumor size (OR 12.08; p <0.0001), nuclear grade (OR 6.99; p <0.0001), pathological stage (OR 35.8; p <0.0001), distance metastasis (OR 4.16; p 0.0001), and death by cancer (OR 4.7; p 0.0004). However, in the multivariate analysis it is not presented as an independent predictor of metastasis (HR 0.45; p 0.11) or cancer-specific mortality (HR 0.93; p 0.91). CONCLUSIONS: In our series, MVI is associated with unfavorable tumors characteristics. In spite of this, it does not seem to be an independent predictor for metastasis and death by non-metastatic renal cancer.
RESUMO
OBJECTIVE: To determine the prognostic impact that tumor size has in patients with pathological renal cancer stage pT3a. METHODS: Retrospective, descriptive study evaluating 261 patients diagnosed with renal cancer pathological stage pT1-3aN0M0 between 1995 and 2013. Clinical and pathological characteristics were evaluated in each group. A ROC curve was used to determine the optimum cutting point of tumor size in relation to the death by cancer. Metastasis-free survival and cancer specific survival were evaluated by the Kaplan Meier method and the differences between the groups were evaluated by the Log Rank test. Multivariate Cox regression analysis was used to evaluate the relationship of tumor size and survival of these patients. RESULTS: 261 patients were studied, 166 of which (63.6%) were Stage pT1a-b, 49 (18.8%) pT2 and 46 (17.6%) pT3a. Patients with pT3a tumors had higher proportion of symptomatic tumors (56.5% vs 33.6% p 0.003), tumor size (7.1 cm vs 5.5 cm; 0.0007), Fuhrman grade 3-4 (52.2% vs 19.1% p 0.0001), coagulative necrosis (62.8% vs 28.8% p 0.0001), distance metastasis (39.1% vs 14.9%; p 0.0001) and death by cancer (23.9% vs 8.9%; p 0.003) when compared with localized tumors (pT1-2). The ROC curve demonstrated that a cut-off point of 7cm is the ideal tumor size to determine renal cancer mortality. Metastasis-free survival at 5 year was 90% for tumors pT1a-b, 71% for pT2, 83% for pT3a <7cm and 48% for pT3a >7cm, with significant statistical differences (Log-rank test <0.001). In the multivariate analysis, only pT3a >7cm stage was an independent predictor of death by renal cancer. CONCLUSIONS: Although perirenal fat invasion and renal vein invasion (pT3a stage) are accepted as prognostic factors, to differentiate this category by tumor size could improve its predictive quality. The tumor diameter (7cm) should be applied to pT3a tumors in order to improve the accuracy of TNM system.
Assuntos
Neoplasias Renais/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Estudos Retrospectivos , Carga TumoralRESUMO
Trypanosoma cruzi is the etiologic agent of Chagas' disease. It is known that amastigotes derived from trypomastigotes in the extracellular milieu are infective in vitro and in vivo. Extracellular amastigotes (EAs) have a stage-specific surface antigen called Ssp-4, a GPI-anchored glycoprotein that is secreted by the parasites. By immunoprecipitation with the Ssp-4-specific monoclonal antibodies (mAb) 2C2 and 1D9, we isolated the glycoprotein from EAs. By mass spectrometry, we identified the core protein of Ssp-4 and evaluated mRNA expression and the presence of Ssp-4 carbohydrate epitopes recognized by mAb1D9. We demonstrated that the carbohydrate epitope recognized by mAb1D9 could promote host cell invasion by EAs. Although infectious EAs express lower amounts of Ssp-4 compared with less-infectious EAs (at the mRNA and protein levels), it is the glycosylation of Ssp-4 (identified by mAb1D9 staining only in infectious strains and recognized by galectin-3 on host cells) that is the determinant of EA invasion of host cells. Furthermore, Ssp-4 is secreted by EAs, either free or associated with parasite vesicles, and can participate in host-cell interactions. The results presented here describe the possible role of a carbohydrate moiety of T. cruzi surface glycoproteins in host cell invasion by EA forms, highlighting the potential of these moieties as therapeutic and vaccine targets for the treatment of Chagas' disease.
RESUMO
Purpose: Diffuse gliomas are the most common primary tumor of the brain and include different subtypes with diverse prognosis. The genomic characterization of diffuse gliomas facilitates their molecular diagnosis. The anatomical localization of diffuse gliomas complicates access to tumor specimens for diagnosis, in some cases incurring high-risk surgical procedures and stereotactic biopsies. Recently, cell-free circulating tumor DNA (ctDNA) has been identified in the cerebrospinal fluid (CSF) of patients with brain malignancies.Experimental Design: We performed an analysis of IDH1, IDH2, TP53, TERT, ATRX, H3F3A, and HIST1H3B gene mutations in two tumor cohorts from The Cancer Genome Atlas (TCGA) including 648 diffuse gliomas. We also performed targeted exome sequencing and droplet digital PCR (ddPCR) analysis of these seven genes in 20 clinical tumor specimens and CSF from glioma patients and performed a histopathologic characterization of the tumors.Results: Analysis of the mutational status of the IDH1, IDH2, TP53, TERT, ATRX, H3F3A, and HIST1H3B genes allowed the classification of 79% of the 648 diffuse gliomas analyzed, into IDH-wild-type glioblastoma, IDH-mutant glioblastoma/diffuse astrocytoma and oligodendroglioma, each subtype exhibiting diverse median overall survival (1.1, 6.7, and 11.2 years, respectively). We developed a sequencing platform to simultaneously and rapidly genotype these seven genes in CSF ctDNA allowing the subclassification of diffuse gliomas.Conclusions: The genomic analysis of IDH1, IDH2, TP53, ATRX, TERT, H3F3A, and HIST1H3B gene mutations in CSF ctDNA facilitates the diagnosis of diffuse gliomas in a timely manner to support the surgical and clinical management of these patients. Clin Cancer Res; 24(12); 2812-9. ©2018 AACR.