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BACKGROUND: Current evidence regarding COVID-19 convalescent plasma (CCP) transfusion practices is limited and heterogeneous. We aimed to determine the impact of the use of CCP transfusion in patients with previous circulating neutralizing antibodies (nAbs) in COVID-19. METHODS: Prospective cohort including 102 patients with COVID-19 transfused with ABO compatible CCP on days 0-2 after enrollment. Clinical status of patients was assessed using the adapted World Health Organization (WHO) ordinal scale on days 0, 5, and 14. The nAbs titration was performed using the cytopathic effect-based virus neutralization test with SARS-CoV-2 (GenBank MT126808.1). The primary outcome was clinical improvement on day 14, defined as a reduction of at least two points on the adapted WHO ordinal scale. Secondary outcomes were the number of intensive care unit (ICU)-free days and the number of invasive mechanical ventilation-free days. RESULTS: Both nAbs of CCP units transfused (p < 0.001) and nAbs of patients before CCP transfusions (p = 0.028) were associated with clinical improvements by day 14. No significant associations between nAbs of patients or CCP units transfused were observed in the number of ICU or mechanical ventilation-free days. Administration of CCP units after 10 days of symptom onset resulted in a decrease in ICU-free days (p < 0.001) and mechanical ventilation-free days (p < 0.001). CONCLUSION: Transfusion of high titer nAbs CCP units may be a determinant in clinical strategies against COVID-19. We consider these data as useful parameters to guide future CCP transfusion practices.
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Anticorpos Neutralizantes/sangue , COVID-19/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Doadores de Sangue , COVID-19/sangue , COVID-19/imunologia , Estudos de Coortes , Feminino , Humanos , Imunização Passiva/métodos , Masculino , Pessoa de Meia-Idade , SARS-CoV-2/imunologia , Soroterapia para COVID-19RESUMO
MOTIVATION: Gene expression data analysis is of great importance for modern molecular biology, given our ability to measure the expression profiles of thousands of genes and enabling studies rooted in systems biology. In this work, we propose a simple statistical model for the activation measuring of gene regulatory networks, instead of the traditional gene co-expression networks. RESULTS: We present the mathematical construction of a statistical procedure for testing hypothesis regarding gene regulatory network activation. The real probability distribution for the test statistic is evaluated by a permutation based study. To illustrate the functionality of the proposed methodology, we also present a simple example based on a small hypothetical network and the activation measuring of two KEGG networks, both based on gene expression data collected from gastric and esophageal samples. The two KEGG networks were also analyzed for a public database, available through NCBI-GEO, presented as Supplementary Material. AVAILABILITY: This method was implemented in an R package that is available at the BioConductor project website under the name maigesPack.
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Redes Reguladoras de Genes , Redes e Vias Metabólicas/genética , Modelos Estatísticos , Bases de Dados Genéticas , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Perfilação da Expressão Gênica/estatística & dados numéricos , Humanos , Modelos Genéticos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologiaRESUMO
A BI-RADS category of 4 from a mammogram indicates suspicious breast lesions, which require core biopsies for diagnosis and have an approximately one third chance of being malignant. Human plasma contains many circulating microRNAs, and variations in their circulating levels have been associated with pathologies, including cancer. Here, we present a novel methodology to identify malignant breast lesions in women with BI-RADS 4 mammography. First, we used the miRNome array and qRT-PCR to define circulating microRNAs that were differentially represented in blood samples from women with breast tumor (BI-RADS 5 or 6) in comparison to controls (BI-RADS 1 or 2). Next, we used qRT-PCR to quantify the level of this circulating microRNAs in patients with mammograms presenting with BI-RADS category 4. Finally, we developed a machine learning method (Artificial Neural Network - ANN) that receives circulating microRNA levels and automatically classifies BI-RADS 4 breast lesions as malignant or benign. We identified a minimum set of three circulating miRNAs (miR-15a, miR-101 and miR-144) with altered levels in patients with breast cancer. These three miRNAs were quantified in plasma from 60 patients presenting biopsy-proven BI-RADS 4 lesions. Finally, we constructed a very efficient ANN that could correctly classify BI-RADS 4 lesions as malignant or benign with approximately 92.5% accuracy, 95% specificity and 88% sensibility. We believe that our strategy of using circulating microRNA and a machine learning method to classify BI-RADS 4 breast lesions is a non-invasive, non-stressful and valuable complementary approach to core biopsy in women with BI-RADS 4 lesions.
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ABSTRACT Laetia suaveolens (Poepp.) Benth., Salicaceae, popularly known as "casinga-cheirosa", "caferana", or "laranjinha", is native to Brazil but not endemic to this country. A crude organic extract was obtained from the leaves and stem and intraperitoneally administered in male Balb-c mice. Its behavioral effects were evaluated in the open field and elevated plus maze in a two-stage experiment that assessed ten different parameters related to behavior as locomotion, emotionality, and anxiety. In the first stage of the experiment, intraperitoneal the crude organic extract administration dose-dependently impaired locomotion and emotionality 30–120 min after administration. A significant decrease in defecation was observed, which was related to emotionality. No alterations in the elevated plus maze were found; thus, this apparatus was not used in the next stage of the experiment. In the second stage, the previously determined non-lethal dose of 0.1563 g/kg was intraperitoneally administered, which impaired locomotion and rearing frequency and increased immobility time. Necropsy revealed smooth intestine hemorrhage. Rutin, leucoside, nicotiflorin, guaijaverin, and astragalin were isolated from the crude organic extract. This is the first time that these compounds have been identified in L. suaveolens. In conclusion, the crude organic extract impaired locomotion and emotionality and caused hemorrhage in male Balb-c mice, indicating that its consumption can be harmful to humans and animals. The present results provide a basis for further studies on the pharmacology, toxicology, and natural product chemistry of the crude organic extract.
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Cancer gene panels (CGPs) are already used in clinical practice to match tumor's genetic profile with available targeted therapies. We aimed to determine if CGPs could also be applied to estimate tumor mutational load and predict clinical benefit to PD-1 and CTLA-4 checkpoint blockade therapy. Whole-exome sequencing (WES) mutation data obtained from melanoma and non-small cell lung cancer (NSCLC) patients published by Snyder et al. 2014 and Rizvi et al. 2015, respectively, were used to select nonsynonymous somatic mutations occurring in genes included in the Foundation Medicine Panel (FM-CGP) and in our own Institutional Panel (HSL-CGP). CGP-mutational load was calculated for each patient using both panels and was associated with clinical outcomes as defined and reported in the original articles. Higher CGP-mutational load was observed in NSCLC patients presenting durable clinical benefit (DCB) to PD-1 blockade (FM-CGP P=0.03, HSL-CGP P=0.01). We also observed that 69% of patients with high CGP-mutational load experienced DCB to PD-1 blockade, as compared to 20% of patients with low CGP-mutational load (FM-CGP and HSL-CGP P=0.01). Noteworthy, predictive accuracy of CGP-mutational load for DCB was not statistically different from that estimated by WES sequencing (P=0.73). Moreover, a high CGP-mutational load was significantly associated with progression-free survival (PFS) in patients treated with PD-1 blockade (FM-CGP P=0.005, HR 0.27, 95% IC 0.105 to 0.669; HSL-CGP P=0.008, HR 0.29, 95% IC 0.116 to 0.719). Similar associations between CGP-mutational load and clinical benefit to CTLA-4 blockade were not observed. In summary, our data reveals that CGPs can be used to estimate mutational load and to predict clinical benefit to PD-1 blockade, with similar accuracy to that reported using WES.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/genética , Análise Mutacional de DNA/métodos , Neoplasias Pulmonares/genética , Melanoma/genética , Área Sob a Curva , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Intervalo Livre de Doença , Perfilação da Expressão Gênica/métodos , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Melanoma/tratamento farmacológico , Melanoma/mortalidade , Mutação , Prognóstico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Curva ROCRESUMO
Laetia suaveolens, known as "casinga-cheirosa", crude extract EB719 has previously shown cytotoxic activity against prostate cancer and squamous cell carcinoma. For the first time, seven molecules were isolated from its apolar-α-tocopherol (1) and sitosterol (2)-and polar-3-O-caffeoylquinic acid (3), 4-O-caffeoylquinic acid (4), 5-O-feruloylquinic acid (5), hyperoside (6), and isoquercitrin (7)-fractions. Acute toxicity was determined in a two-stage experiment: (1) a reduced number of Balb-c male mice received 5000 mg/kg of EB719 to allow evaluation of general activity and other 27 parameters, plus death, up to the establishment of non-lethal dose (NLD), as well as lethal dose 50% (LD50); (2) NLD was administered and diazepam introduced as reference drug. EB719 showed LD50=178.0 mg/kg, and NLD 156.3 mg/kg. In stage one EB719 did not influence general activity, but provoked impairment in grasp reflexes, tail squeeze and breathing; piloerection and cyanosis were increased. In stage two, alterations occurred in auricular reflex, piloerection and breathing after diazepam administration, but not in response to EB719. Intestinal hemorrhage caused by local bleeding was observed after necropsy, and may be the main cause of animals' death other than a systemic effect of the extract. Although the isolated compounds are biologically and pharmacologically active in both men and animal systems, it is premature to relate their occurrence in EB719 to the observed intestine hemorrhage in mice.
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Hemorragia Gastrointestinal/induzido quimicamente , Extratos Vegetais/toxicidade , Salicaceae/química , Animais , Peso Corporal , Diazepam/toxicidade , Hemorragia Gastrointestinal/patologia , Humanos , Dose Letal Mediana , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Tamanho do Órgão , Piloereção/efeitos dos fármacos , Extratos Vegetais/química , Quercetina/análogos & derivados , Quercetina/isolamento & purificação , Ácido Quínico/análogos & derivados , Ácido Quínico/isolamento & purificação , Respiração/efeitos dos fármacos , Sitosteroides/isolamento & purificação , alfa-Tocoferol/isolamento & purificaçãoRESUMO
BACKGROUND: Cutaneous melanoma displays high morbidity and mortality rates. Isolated limb perfusion with melphalan (Mel) is used for the treatment of non-resectable, locally advanced extremity melanomas. When combined with tumor necrosis factor alpha (TNF-alpha) treatment, the complete response varies between 70% and 90%. The mechanisms underlying the effects of Mel and TNF-alpha are not completely understood. We evaluated the impact of systemic Mel and TNF-alpha administration on tumor growth, analyzed the morphological changes promoted by each treatment, and identified early expressed genes in response to Mel and TNF-alpha treatment, either alone or in combination, in a murine melanoma model. METHODS: Six- to eight-week-old male mice were subcutaneously inoculated with B16F10 melanoma cells and then intravenously injected with TNF-alpha, melphalan or a combination of both drugs when the tumors reached 1.0 cm(2). Tumor growth was monitored every other day, and histological analysis was performed when the tumors reached 3.0 cm(2). Total RNA was extracted from the resected tumors and submitted to amplification, labeling and hybridization on an oligonucleotide microarray (Fox Chase Cancer Center). Tumor growth and histological parameters were compared using ANOVA. Survival curves were calculated using the Kaplan-Meier method. Two-way ANOVA was used to identify differentially expressed genes among the various treatments, and Dunn's test was used for pair-wise comparisons. RESULTS: Systemic administration of Mel impaired tumor growth (p<0.001), improved animal survival (p<0.001), and decreased mitotic rate (p=0.049). Treatment with TNF-alpha alone had no impact, neither on tumor growth, nor on survival, but it increased necrosis (p<0.024) and decreased mitotic rates (p=0.001) in the tumors. Combined treatment with Mel and TNF-alpha had similar effects in tumor growth, survival, necrosis and mitotic rate as observed with individual treatments. Moreover, 118 genes were found differentially expressed by microarray analysis and 10% of them were validated by RT- real time PCR. In our model we found that the treatments regulate genes that play important roles in tumorigenesis such as cell adhesion (Pard3, Pecam1, Ilk, and Dlg5), proliferation (Tcfe3 and Polr1e), cell motility (Kifap3, Palld, and Arhgef6), apoptosis (Bcl2l11), and angiogenesis (Flt1 and Ptprj). CONCLUSIONS: Our data reproduces, in mice, some of the features observed in melanoma patients treated with the combination of Mel and TNF-alpha. The identification of genes with altered expression by these drugs both individually and in combination might help in the understanding of their mechanism of action and, as a consequence, improved strategies that could impact their clinical application.
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Antineoplásicos Alquilantes/uso terapêutico , Melanoma/tratamento farmacológico , Melfalan/uso terapêutico , Fator de Necrose Tumoral alfa/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Masculino , Melanoma/genética , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neovascularização Patológica/genética , Resultado do TratamentoRESUMO
BACKGROUND: Topoisomerase 2 alpha (TOP2A), HER-2/neu, and survivin are genes that lie on chromosome 17 and correlate with the prognosis and prediction of target-driven therapy against tumors. In a previous study, we showed that TOP2A transcripts levels were significantly higher in soft tissue sarcomas (STS) than in benign tumors and desmoid-type fibromatoses (FM). Because these genes have been insufficiently examined in STS, we aimed to identify alterations in TOP2A and HER-2 expression by fluorescent in situ hybridization and immunohistochemistry, as well as that of survivin, and correlate them with clinicopathologic findings to assess their prognostic value. METHODS: Eighteen FM and 244 STS were included. Fluorescent in situ hybridization and immunohistochemistry were performed on a tissue microarray. RESULTS: TOP2A and survivin were more highly expressed in sarcomas than in FM. TOP2A was an independent predictor of an unfavorable prognosis; it was combined with formerly established prognostic factors (primarily histologic grade and tumor size at diagnosis) to create a prognostic index that evaluated overall survival. Gene amplification/polysomy (13%) did not correlate with protein overexpression. Survivin and HER-2 expression were not associated with patient outcomes. CONCLUSIONS: These findings might become valuable in the management of patients with STS and possibly in the prospective evaluation of responses to new target-driven therapies.
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Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Cromossomos Humanos Par 17/genética , Amplificação de Genes , Sarcoma/mortalidade , Sarcoma/patologia , Adolescente , Adulto , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Fibroma/mortalidade , Fibroma/patologia , Fibroma/terapia , Fibromatose Agressiva/mortalidade , Fibromatose Agressiva/patologia , Fibromatose Agressiva/terapia , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Hibridização in Situ Fluorescente , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Proteínas de Ligação a Poli-ADP-Ribose , Prognóstico , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Sarcoma/terapia , Sarcoma Sinovial/mortalidade , Sarcoma Sinovial/patologia , Sarcoma Sinovial/terapia , Taxa de Sobrevida , Survivina , Adulto JovemRESUMO
BACKGROUND: Cancer shows a great diversity in its clinical behavior which cannot be easily predicted using the currently available clinical or pathological markers. The identification of pathways associated with lymph node metastasis (N+) and recurrent head and neck squamous cell carcinoma (HNSCC) may increase our understanding of the complex biology of this disease. METHODS: Tumor samples were obtained from untreated HNSCC patients undergoing surgery. Patients were classified according to pathologic lymph node status (positive or negative) or tumor recurrence (recurrent or non-recurrent tumor) after treatment (surgery with neck dissection followed by radiotherapy). Using microarray gene expression, we screened tumor samples according to modules comprised by genes in the same pathway or functional category. RESULTS: The most frequent alterations were the repression of modules in negative lymph node (N0) and in non-recurrent tumors rather than induction of modules in N+ or in recurrent tumors. N0 tumors showed repression of modules that contain cell survival genes and in non-recurrent tumors cell-cell signaling and extracellular region modules were repressed. CONCLUSIONS: The repression of modules that contain cell survival genes in N0 tumors reinforces the important role that apoptosis plays in the regulation of metastasis. In addition, because tumor samples used here were not microdissected, tumor gene expression data are represented together with the stroma, which may reveal signaling between the microenvironment and tumor cells. For instance, in non-recurrent tumors, extracellular region module was repressed, indicating that the stroma and tumor cells may have fewer interactions, which disable metastasis development. Finally, the genes highlighted in our analysis can be implicated in more than one pathway or characteristic, suggesting that therapeutic approaches to prevent tumor progression should target more than one gene or pathway, specially apoptosis and interactions between tumor cells and the stroma.
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Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Progressão da Doença , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Transdução de Sinais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/diagnóstico , Sobrevivência Celular/genética , Feminino , Perfilação da Expressão Gênica , Neoplasias de Cabeça e Pescoço/diagnóstico , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Prognóstico , Células Estromais/patologia , Adulto JovemRESUMO
Sequencing technologies and new bioinformatics tools have led to the complete sequencing of various genomes. However, information regarding the human transcriptome and its annotation is yet to be completed. The Human Cancer Genome Project, using ORESTES (open reading frame EST sequences) methodology, contributed to this objective by generating data from about 1.2 million expressed sequence tags. Approximately 30% of these sequences did not align to ESTs in the public databases and were considered no-match ORESTES. On the basis that a set of these ESTs could represent new transcripts, we constructed a cDNA microarray. This platform was used to hybridize against 12 different normal or tumor tissues. We identified 3421 transcribed regions not associated with annotated transcripts, representing 83.3% of the platform. The total number of differentially expressed sequences was 1007. Also, 28% of analyzed sequences could represent noncoding RNAs. Our data reinforces the knowledge of the human genome being pervasively transcribed, and point out molecular marker candidates for different cancers. To reinforce our data, we confirmed, by real-time PCR, the differential expression of three out of eight potentially tumor markers in prostate tissues. Lists of 1007 differentially expressed sequences, and the 291 potentially noncoding tumor markers were provided.
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Biomarcadores Tumorais/biossíntese , Etiquetas de Sequências Expressas , RNA não Traduzido/biossíntese , Biomarcadores Tumorais/genética , Mapeamento Cromossômico , Etiquetas de Sequências Expressas/química , Perfilação da Expressão Gênica , Genoma Humano , Genômica , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , RNA Neoplásico/biossíntese , Transcrição GênicaRESUMO
Biosafety of genetically modified organisms (GMOs) and their derivatives is still a major topic in the agenda of government and societies worldwide. The aim of this review is to bring into light that data that supported the decision taken back in 1998 as an exercise to stimulate criticism from the scientific community for upcoming discussions and to avoid emotional and senseless arguments that could jeopardize future development in the field. It must be emphasized that Roundup Ready soybean is just one example of how biotechnology can bring in significant advances for society, not only through increased productivity, but also with beneficial environmental impact, thereby allowing more rational use of agricultural pesticides for improvement of the soil conditions. The adoption of agricultural practices with higher yield will also allow better distribution of income among small farmers. New species of genetically modified plants will soon be available and society should be capable of making decisions in an objective and well-informed manner, through collegiate bodies that are qualified in all aspects of biosafety and environmental impact.
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3-Fosfoshikimato 1-Carboxiviniltransferase/genética , Alimentos Geneticamente Modificados , Glycine max/genética , Plantas Geneticamente Modificadas/genética , 3-Fosfoshikimato 1-Carboxiviniltransferase/análise , 3-Fosfoshikimato 1-Carboxiviniltransferase/química , Animais , Brasil , Qualidade de Produtos para o Consumidor/legislação & jurisprudência , Humanos , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/toxicidadeRESUMO
Biosafety of genetically modified organisms (GMOs) and their derivatives is still a major topic in the agenda of government and societies worldwide. The aim of this review is to bring into light that data that supported the decision taken back in 1998 as an exercise to stimulate criticism from the scientific community for upcoming discussions and to avoid emotional and senseless arguments that could jeopardize future development in the field. It must be emphasized that Roundup Ready® soybean is just one example of how biotechnology can bring in significant advances for society, not only through increased productivity, but also with beneficial environmental impact, thereby allowing more rational use of agricultural pesticides for improvement of the soil conditions. The adoption of agricultural practices with higher yield will also allow better distribution of income among small farmers. New species of genetically modified plants will soon be available and society should be capable of making decisions in an objective and well-informed manner, through collegiate bodies that are qualified in all aspects of biosafety and environmental impact.
A biosegurança dos organismos geneticamente modificados e seus derivados é um dos principais tópicos na agenda de discussões de governos e sociedades. O objetivo desta revisão é reviver os dados científicos que fundamentaram a decisão de liberação comercial da soja transgênica resistente ao Glifosate com o intuito de estimular uma posição crítica da comunidade científica para as próximas discussões no tema. A soja em questão é apenas um exemplo de como a biotecnologia pode contribuir para avanços na produtividade e na preservação do meio ambiente, com ganho de produtividade e lucratividade para agricultores em todas as escalas. Novas variedades trangênicas estarão na pauta de discussões que deverão estar fundamentadas em dados científicos objetivos, evitando argumentos emocionais que poderão, assim como no passado recente, prejudicar o desenvolvimento científico e tecnológico da agricultura.
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Animais , Humanos , /genética , Alimentos Geneticamente Modificados , Plantas Geneticamente Modificadas/genética , Glycine max/genética , /análise , /química , Brasil , Qualidade de Produtos para o Consumidor/legislação & jurisprudência , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/toxicidadeRESUMO
C57BL/6 and BALB/c mice are prototype hosts for the study of resistance and susceptibility to several infectious diseases. In many cases, resistance of C57BL/6 is due to the microbicidal effect of nitric oxide (NO) produced by macrophages in response to interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), mainly secreted by Th1 cells and macrophages, respectively. BALB/c, usually unable to give rise to Th1 lymphocytes, does not control certain infections. However, we and others have previously observed that regardless of the adaptive immune response, C57BL/6 (M-1) macrophages are far more sensitive to the stimulus of IFN-gamma-plus lipopolysaccharide (LPS) for the production of NO than are BALB/c (M-2) cells, a feature that might also account for resistance. Here, we report that the differential production of NO by M-1 and M-2 macrophages correlates with the accumulation of inducible nitric oxide synthase (iNOS) mRNA and protein, which shows that expression of iNOS is differentially regulated in M-1 and M-2 cells. The higher accumulation of iNOS mRNA in M-1 cells is independent of its stability, and, thus, it is possible that transcription of the iNOS gene in these cells may be more efficient than in M-2 cells. A remarkable finding is that the level of iNOS protein is much higher in M-1 macrophages than in M-2 cells, as compared with the mRNA levels, which makes us speculate that differential translational or posttranslational controls of iNOS gene are operative.
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Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Animais , Células Cultivadas , Interferon gama/imunologia , Ativação de Macrófagos , Macrófagos Peritoneais/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/genética , Estabilidade de RNA , Fatores de TempoRESUMO
Adenocarcinomas of stomach and esophagus are frequently associated with preceding inflammatory alterations of the normal mucosa. Whereas intestinal metaplasia of the gastric mucosa is associated with higher risk of malignization, Barrett's disease is a risk factor for adenocarcinoma of the esophagus. Barrett's disease is characterized by the substitution of the squamous mucosa of the esophagus by a columnar tissue classified histopathologically as intestinal metaplasia. Using cDNA microarrays, we determined the expression profile of normal gastric and esophageal mucosa as well as intestinal metaplasia and adenocarcinomas from both organs. Data were explored to define functional alterations related to the transformation from squamous to columnar epithelium and the malignant transformation from intestinal metaplasia to adenocarcinomas. Based on their expression profile, adenocarcinomas of the esophagus showed stronger correlation with intestinal metaplasia of the stomach than with Barrett's mucosa. Second, we identified two functional modules, lipid metabolism and cytokine, as being altered with higher statistical significance. Whereas the lipid metabolism module is active in samples representing intestinal metaplasia and inactive in adenocarcinomas, the cytokine module is inactive in samples representing normal esophagus and esophagitis. Using the concept of relevance networks, we determined the changes in linear correlation of genes pertaining to these two functional modules. Exploitation of the data presented herein will help in the precise molecular characterization of adenocarcinoma from the distal esophagus, avoiding the topographical and descriptive classification that is currently adopted, and help with the proper management of patients with Barrett's disease.
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Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Metabolismo dos Lipídeos , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Adenocarcinoma/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Citocinas/biossíntese , Citocinas/genética , Citocinas/metabolismo , Neoplasias Esofágicas/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Gástricas/patologiaRESUMO
Using cDNA microarrays with 3800 cDNA fragments, we determined the expression profile of normal thyroid tissue, goiter, adenoma and papillary carcinoma (10 samples from each class). After background correction and statistical analysis, we identified a set of 160 genes as being differentially expressed in all pair-wise comparisons. Here we demonstrate that, at least on the basis of these differentially expressed genes, a positive correlation between goiter and papillary carcinomas could be observed. We identified a common set of genes whose expression is diminished in both goiter and papillary carcinomas as compared to normal thyroid tissue. Moreover, no genes with inverse correlation in samples from goiter and papillary carcinomas could be detected. Using Real-Time PCR and/or tissue microarrays, we confirmed the altered expression of some of the identified genes. Of notice, we demonstrate that the reduced mRNA levels of p27(kip1) observed in papillary carcinomas as compared to either goiter or normal thyroid tissues (P<0.001) is accompanied by an altered protein distribution within the cell. In papillary carcinomas, P27(KIP1) is preferentially cytoplasmic as opposed to goiter or normal thyroid tissue, where P27(KIP1) is preferentially located in the nucleus. The exploitation of the data presented here could contribute to the understanding of the molecular events related to thyroid diseases and gives support to the notion that common molecular events might be related to the frequent observation of areas of papillary carcinomas in the gland of patients with goiter.
Assuntos
Carcinoma Papilar/genética , Perfilação da Expressão Gênica , Bócio/genética , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genética , Proteínas de Transporte/análise , Inibidor de Quinase Dependente de Ciclina p27 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise Serial de TecidosRESUMO
During the last 5 years, the number of papers describing data obtained by microarray technology increased exponentially with about 3000 papers in 2003. Undoubtedly, cancer is by far the disease that received most of the attention as far as the amount of data generated. As array technology is rather new and highly dependent on bioinformatics, mathematics and statistics, a clear understanding of the knowledge and information derived from array-based experiments is not widely appreciated. We shall review herein some of the issues related to the construction of DNA arrays, quantities and heterogeneity of probes and targets, the consequences of the physical characteristics of the probes, data extraction and data analysis as well as the applications of array technology. Our goal is to bring to the general audience, some of the basics of array technology and its possible application in oncology. By discussing some of the basic aspects of the methodology, we hope to stimulate criticism concerning the conclusions proposed by authors, especially in the light of the very low degree of reproducibility already proven when commercially available platforms were compared . Regardless of its pitfalls, it is unquestionable that array technology will have a great impact in the management of cancer and its applications will range from the discovery of new drug targets, new molecular tools for diagnosis and prognosis as well as for a tailored treatment that will take into account the molecular determinants of a given tumor. Hence, we shall also highlight some of the already available and promising applications of array technology on the day-to-day practice of oncology.
Assuntos
Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise por Conglomerados , Biologia Computacional , Humanos , Neoplasias/diagnóstico , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , RNA Neoplásico/análise , RNA Neoplásico/genéticaRESUMO
We studied bone marrow stromal cell cultures from patients with childhood myelodysplastic syndromes (MDS, refractory anemia with excess of blasts, RAEB) and from matched normal donors. Stromal cell monolayers were characterized as myofibroblasts by the expression of smooth muscle alpha-actin, collagen IV, laminin and fibronectin. When normal cord blood cells were plated onto myelodysplastic stromas, a pathologic cell differentiation was observed, indicating altered myelosupportive properties. cDNA array analysis showed that patient stromas expressed increased levels of thrombospondin-1, collagen-I alpha2-chain, osteoblast-specific factor-2 and osteonectin, indicating the presence of increased osteoblast content, as confirmed by enhanced alkaline phosphatase synthesis. Alterations in the myelodysplastic stroma environment might contribute to abnormal hematopoiesis in this pathology.
Assuntos
Medula Óssea/patologia , Regulação Neoplásica da Expressão Gênica , Hematopoese , Músculo Liso/patologia , Síndromes Mielodisplásicas/patologia , Células Estromais/patologia , Actinas/metabolismo , Fosfatase Alcalina/metabolismo , Anemia Refratária com Excesso de Blastos , Medula Óssea/metabolismo , Estudos de Casos e Controles , Diferenciação Celular , Criança , Pré-Escolar , Colágeno Tipo IV/metabolismo , Feminino , Sangue Fetal/química , Sangue Fetal/metabolismo , Fibroblastos/patologia , Fibronectinas/metabolismo , Perfilação da Expressão Gênica , Humanos , Técnicas In Vitro , Lactente , Laminina/metabolismo , Masculino , Músculo Liso/metabolismo , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Osteoblastos/metabolismo , Pré-Leucemia , Células Estromais/metabolismoRESUMO
We have previously demonstrated that Staphylococcus aureus, a highly invasive bacteria, presents a 52-kDa surface protein that mediates its binding to laminin. In order to better characterize this receptor, we excised this putative laminin receptor from two-dimensional (2-D) PAGE and used it as antigen for raising a mouse hyperimmune serum which was for screening an S. aureus expression library. A single clone of 0.3 kb was obtained, and its sequence revealed 100% homology with S. aureus alpha-enolase. Moreover, amino acid sequencing of the 52-kDa protein eluted from the 2-D gel indicated its molecular homology with alpha-enolase, an enzyme that presents a high evolutionary conservation among species. In parallel, monoclonal antibodies raised against the S. aureus 52-kDa band also recognized yeast alpha-enolase in western blot analysis. These monoclonal antibodies were also able to promote capture of iodine-labeled bacteria when adsorbed to a solid phase, and this capture was inhibited by the addition of excess rabbit muscle alpha-enolase. Finally, the cell surface localization of S. aureus alpha-enolase was further confirmed by flow cytometry. Hence, alpha-enolase might play a critical role in the pathogenesis of S. aureus by allowing its adherence to laminin-containing extracellular matrix.
Assuntos
Laminina/metabolismo , Fosfopiruvato Hidratase/metabolismo , Receptores de Laminina/metabolismo , Staphylococcus aureus/enzimologia , Aderência Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Biblioteca Gênica , Genes Bacterianos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfopiruvato Hidratase/química , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/imunologia , Ligação Proteica , Receptores de Laminina/química , Receptores de Laminina/imunologia , Análise de Sequência de DNA , Análise de Sequência de Proteína , Homologia de Sequência , Staphylococcus aureus/patogenicidadeAssuntos
Masculino , Feminino , Humanos , DNA , Genoma , Biologia Molecular , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiologia , Fatores de RiscoRESUMO
High incidence of gastric cancer-related death is mainly due to diagnosis at an advanced stage in addition to the lack of adequate neoadjuvant therapy. Hence, new tools aimed at early diagnosis would have a positive impact in the outcome of the disease. Using cDNA arrays having 376 genes either identified previously as altered in gastric tumors or known to be altered in human cancer, we determined expression signature of 99 tissue fragments representing normal gastric mucosa, gastritis, intestinal metaplasia, and adenocarcinomas. We first validated the array by identifying molecular markers that are associated with intestinal metaplasia, considered as a transition stage of gastric adenocarcinomas of the intestinal type as well as markers that are associated with diffuse type of gastric adenocarcinomas. Next, we applied Fisher's linear discriminant analysis in an exhaustive search of trios of genes that could be used to build classifiers for class distinction. Many classifiers could distinguish between normal and tumor samples, whereas, for the distinction of gastritis from tumor and for metaplasia from tumor, fewer classifiers were identified. Statistical validations showed that trios that discriminate between normal and tumor samples are powerful classifiers to distinguish between tumor and nontumor samples. More relevant, it was possible to identify samples of intestinal metaplasia that have expression signature resembling that of an adenocarcinoma and can now be used for follow-up of patients to determine their potential as a prognostic test for malignant transformation.