RESUMO
In Plasmodium, the first two and rate-limiting enzymes of the pentose phosphate pathway, glucose 6-phosphate dehydrogenase (G6PD) and the 6-phosphogluconolactonase, are bifunctionally fused to a unique enzyme named GluPho, differing structurally and mechanistically from the respective human orthologs. Consistent with the enzyme's essentiality for malaria parasite proliferation and propagation, human G6PD deficiency has immense impact on protection against severe malaria, making PfGluPho an attractive antimalarial drug target. Herein we report on the optimized lead compound N-(((2R,4S)-1-cyclobutyl-4-hydroxypyrrolidin-2-yl)methyl)-6-fluoro-4-methyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxamide (SBI-0797750), a potent and fully selective PfGluPho inhibitor with robust nanomolar activity against recombinant PfGluPho, PvG6PD, and P. falciparum blood-stage parasites. Mode-of-action studies have confirmed that SBI-0797750 disturbs the cytosolic glutathione-dependent redox potential, as well as the cytosolic and mitochondrial H2O2 homeostasis of P. falciparum blood stages, at low nanomolar concentrations. Moreover, SBI-0797750 does not harm red blood cell (RBC) integrity and phagocytosis and thus does not promote anemia. SBI-0797750 is therefore a very promising antimalarial lead compound.
Assuntos
Antimaláricos , Deficiência de Glucosefosfato Desidrogenase , Malária Falciparum , Malária Vivax , Malária , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Hidrolases de Éster Carboxílico , Glucose/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Malária Falciparum/tratamento farmacológico , Malária Vivax/tratamento farmacológico , Fosfatos , Plasmodium falciparum/metabolismo , Plasmodium vivaxAssuntos
Eritrócitos/metabolismo , Deficiência de Glucosefosfato Desidrogenase/metabolismo , Glucosídeos/farmacologia , Hemólise/efeitos dos fármacos , Pirimidinonas/farmacologia , Sementes/química , Vicia faba/química , Adulto , Criança , Pré-Escolar , Eritrócitos/patologia , Feminino , Deficiência de Glucosefosfato Desidrogenase/patologia , Glucosídeos/química , Humanos , Masculino , Pirimidinonas/químicaRESUMO
BACKGROUND: While G6PD deficiency is one of the major causes of acute hemolytic anemia, the membrane changes leading to red cell lysis have not been extensively studied. New findings concerning the mechanisms of G6PD deficient red cell destruction may facilitate our understanding of the large individual variations in susceptibility to pro-oxidant compounds and aid the prediction of the hemolytic activity of new drugs. METHODOLOGY/PRINCIPAL FINDINGS: Our results show that treatment of G6PD deficient red cells with diamide (0.25 mM) or divicine (0.5 mM) causes: (1) an increase in the oxidation and tyrosine phosphorylation of AE1; (2) progressive recruitment of phosphorylated AE1 in large membrane complexes which also contain hemichromes; (3) parallel red cell lysis and a massive release of vesicles containing hemichromes. We have observed that inhibition of AE1 phosphorylation by Syk kinase inhibitors prevented its clustering and the membrane vesiculation while increases in AE1 phosphorylation by tyrosine phosphatase inhibitors increased both red cell lysis and vesiculation rates. In control RBCs we observed only transient AE1 phosphorylation. CONCLUSIONS/SIGNIFICANCE: Collectively, our findings indicate that persistent tyrosine phosphorylation produces extensive membrane destabilization leading to the loss of vesicles which contain hemichromes. The proposed mechanism of hemolysis may be applied to other hemolytic diseases characterized by the accumulation of hemoglobin denaturation products.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Membrana Eritrocítica/patologia , Deficiência de Glucosefosfato Desidrogenase/patologia , Proteínas Tirosina Quinases/metabolismo , Membrana Eritrocítica/ultraestrutura , Hemoglobinas/metabolismo , Hemólise , Humanos , Oxidantes/farmacologia , Fosforilação/efeitos dos fármacos , Tirosina/metabolismoRESUMO
A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method for detection of cytokeratin 20-positive cells in blood characterized by two novel features was developed and tested on 99 patients with colorectal cancer, 110 with breast cancer, and 150 healthy subjects. To optimize the specificity and sensitivity of the method, two novel features were used. First, a primer overlapping two adjacent exons was generated to inhibit nonspecific amplification both in healthy donors and cancer patients; second, a non-end-point first-round amplification was used to increase sensitivity. The number of first-round cycles was chosen to reach the highest level of sensitivity while conserving quantitative characteristics. PCR efficiency increased from 88.9% in single-round RT-PCR to 99.0% in nested real-time RT-PCR. To establish sensitivity and specificity of the method, HT29 cells were serially diluted with normal blood. Detection limit improved from 100 HT29 cells (single-round RT-PCR) to 1 to 10 cells (nested real-time RT-PCR) per 3 ml of whole blood. None of the healthy subjects was positive, whereas 22 and 29% of all colorectal and breast cancer patients, respectively, had cytokeratin 20 cell equivalents in blood. The association between cytokeratin 20 cell equivalents and metastasis was statistically significant for breast (P = 0.026) but not colorectal cancer patients (P = 0.361). Negativity of all 150 healthy controls examined confers diagnostic potential to the method.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Neoplasias Colorretais/metabolismo , Queratinas/sangue , RNA Neoplásico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Neoplasias da Mama/diagnóstico , Neoplasias Colorretais/diagnóstico , Humanos , Queratina-20 , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
BACKGROUND: A number of noninvasive tests have been developed to establish the presence of Helicobacter pylori infection. Although polyclonal antibody-based stool antigen testing has a good sensitivity and specificity, it is less accurate than urea breath testing. Recently, a monoclonal antibody-based stool antigen test demonstrated an excellent performance in diagnosing H. pylori infection in adults and in pediatric populations. AIM: To evaluate the diagnostic accuracy of a novel stool test based on monoclonal antibodies to detect H. pylori antigens in frozen human stool in the pretreatment setting. PATIENTS AND METHODS: Stool specimens were prospectively collected from 78 patients undergoing gastroscopy and stored at -20 degrees C until tested. Helicobacter pylori infection was evaluated by histology, rapid urease testing and urea breath tests ((13)C-UBT). Positivity of the three tests was considered the gold standard for H. pylori active infection. Patients with no positive test were considered negative. The gold standard was compare to the results of the monoclonal antibody stool antigen test. Frozen stool specimens were tested using a novel monoclonal-antibody-based enzyme immunoassay (HePy-Stool, Biolife-Italiana, Milan, Italy). RESULTS: The sensitivity and specificity of the monoclonal stool antigen test were 97%[95% confidence interval, (CI) 86-100] and 94% (95% CI: 81-99), respectively. Negative and positive predictive values were 97% (95% CI: 85-99), and 95% (95% CI: 83-99), respectively. The diagnostic accuracy was 96% (95% CI: 88-99). The likelihood ratio for a positive test was 17 and for a negative test was 0. CONCLUSIONS: Although the (13)C-UBT is the most accurate among the available noninvasive tests, our results show that an H. pylori stool test using monoclonal antibody might be an excellent alternative.
Assuntos
Anticorpos Antibacterianos , Anticorpos Monoclonais , Antígenos de Bactérias/análise , Fezes/microbiologia , Infecções por Helicobacter/diagnóstico , Helicobacter pylori/isolamento & purificação , Adulto , Idoso , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Feminino , Infecções por Helicobacter/microbiologia , Helicobacter pylori/imunologia , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Chronic infections have been associated with cardiovascular disease. We used bacterial culture, polymerase chain reaction (PCR), and immunohistochemical staining with anti-vacA and anticagA antibodies to search for Helicobacter pylori and Chlamydiae pneumoniae in atherosclerotic plaques obtained at endarterectomy. Serum IgG antibodies to H. pylori and C. pneumoniae were also determined. Thirty-two patients were enrolled. Anti-H. pylori and anti-C. pneumoniae IgG were present in 72% and 81%, respectively. Culture and PCR for H. pylori of vessel walls and plaques were negative. Atherosclerotic plaque and normal vessel sections from H. pylori-negative and- positive patients showed reactivity with anti-vacA and anti-cagA antibodies. C. pneumoniae DNA was amplified in three atherosclerotic lesions. These findings suggest that the association between H. pylori infection and atherosclerosis does not result from continuing direct effects of H. pylori antigens in the vessel walls. Antigens within vessel atherosclerotic plaques cross-react with H. pylori virulence factors and could act as cofactors in determining instability for the atherosclerotic plaques.