Trombolisis en el tromboembolismo de pulmón

Fil: Valdivieso, W.. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Área de CardiologíaFil: Gambarte, A.. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Área de CardiologíaFil: Piasentín, J.. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Área de CardiologíaFil: Limia, P.. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Área de Cardiologí

Circulating galectin-1 delineates response to bevacizumab in melanoma patients and reprograms endothelial cell biology

Blockade of vascular endothelial growth factor (VEGF) signaling with bevacizumab, a humanized anti-VEGF monoclonal antibody (mAb), or with receptor tyrosine kinase inhibitors, has improved progression-free survival and, in some indications, overall survival across several types of cancers by interrupting tumor angiogenesis. However, the clinical benefit conferred by these therapies is variable, and tumors from treated patients eventually reinitiate growth. Previously we demonstrated, in mouse tumor models, that galectin-1 (Gal1), an endogenous glycan-binding protein, preserves angiogenesis in anti-VEGF–resistant tumors by co-opting the VEGF receptor (VEGFR)2 signaling pathway in the absence of VEGF. However, the relevance of these findings in clinical settings is uncertain. Here, we explored, in a cohort of melanoma patients from AVAST-M, a multicenter, open-label, randomized controlled phase 3 trial of adjuvant bevacizumab versus standard surveillance, the role of circulating plasma Gal1 as part of a compensatory mechanism that orchestrates endothelial cell programs in bevacizumab-treated melanoma patients. We found that increasing Gal1 levels over time in patients in the bevacizumab arm, but not in the observation arm, significantly increased their risks of recurrence and death. Remarkably, plasma Gal1 was functionally active as it was able to reprogram endothelial cell biology, promoting migration, tubulogenesis, and VEGFR2 phosphorylation. These effects were prevented by blockade of Gal1 using a newly developed fully human anti-Gal1 neutralizing mAb. Thus, using samples from a large-scale clinical trial from stage II and III melanoma patients, we validated the clinical relevance of Gal1 as a potential mechanism of resistance to bevacizumab treatment

Optimization of the in vitro comet assay as a tool for mechanistic risk assessment

Genotoxicity evaluation is of key importance in the health risk assessment of substances to which humans are exposed, as it has been long established that certain genotoxic compounds are able to damage DNA entailing severe consequences for human health, such as cancer. Current strategies of genotoxicity testing consider mainly final effects in DNA: mutations and chromosomal aberrations. However, a mechanistic approach more relevant to humans, in which not only classical endpoints but also mechanistic events (e.g., DNA oxidation or alkylation) are integrated and considered for risk assessment, is becoming more necessary. In this context, modifications to the in vitro comet assay protocol, which measures strand breaks and alkali labile sites in its standard version, arise as a promising alternative method in the detection of premutagenic lesions. The aim of this thesis was to develop and validate a new tool for in vitro genotoxicity testing, based on the comet assay, that can be used in the elucidation of different mechanisms of action. This approach may represent a good candidate for complementing current in vitro genotoxicity testing batteries. The combination of the comet assay with lesion-specific enzymes is used to detect altered bases. A review about this version of the assay revealed that formamidopyrimidine DNA glycosylase (Fpg) is the most used enzyme, used for the detection of oxidized bases. In total, 12 different enzymes have been combined with the comet assay to detect other lesions such as alkylated bases, presence of uracil, pyrimidine dimers or AP-sites. The areas of application in which the enzyme-modified comet assay has been more extensively used are in vitro genotoxicity testing and human biomonitoring. For the detection of alkylated bases, two non-commercially available bacterial enzymes, 3-methyladenine DNA glycosylase II (AlkA) and 3-methyladenine DNA glycosylase (AlkD), have been sporadically employed. In this thesis, a commercial human alkyladenine DNA glycosilase (hAAG) was successfully applied for the first time. Moreover, the use of hAAG together with other different enzymes (non-commercial Fpg and commercial Fpg, Endonuclease III -Endo III- and human 8-oxoguanine DNA glycosylase -hOGG1-), with various specificities towards oxidized lesions, was optimized to be used on a single assay using a medium throughput format (i.e., 12 minigels/slide). To this aim, the incubation conditions when using the widely used 2 gels/slide format and the medium-throughput 12 minigels/slide format was assessed. This comparison highlighted that is crucial to perform enzyme titration experiments using the same protocol, equipment and the format that are going to be used in the final experiments. Moreover, in order to detect DNA cross-links, an extra DNA lesion which may be difficult to detect by mean of enzymes, an already known modification of the comet assay was set up using the same throughput format. Finally, both comet assay modifications were validated using TK-6 cells treated with non-cytotoxic concentrations of nine compounds with several mechanisms of action: oxidizing and alkylating agents, cross-linkers, a bulky-adducts inducer and non-genotoxic compounds. The combination of the results of both modifications allowed to clearly differentiate the induced lesions, with the exception of the bulky adducts, which was expected. Moreover, no DNA lesions was detected in cells treated with the non-genotoxic compounds. Both Fpg enzymes (non-commercial and commercial one) gave same results. The in vitro comet assay modified with several enzymes together with the cross-links modification increases significantly the comet assay ability to detect different premutagenic lesions, providing genotoxic mechanistic information about the type of damage. Its application might be very relevant in the current regulatory context

Optimization of the in vitro comet assay as a tool for mechanistic risk assessment

Genotoxicity evaluation is of key importance in the health risk assessment of substances to which humans are exposed, as it has been long established that certain genotoxic compounds are able to damage DNA entailing severe consequences for human health, such as cancer. Current strategies of genotoxicity testing consider mainly final effects in DNA: mutations and chromosomal aberrations. However, a mechanistic approach more relevant to humans, in which not only classical endpoints but also mechanistic events (e.g., DNA oxidation or alkylation) are integrated and considered for risk assessment, is becoming more necessary. In this context, modifications to the in vitro comet assay protocol, which measures strand breaks and alkali labile sites in its standard version, arise as a promising alternative method in the detection of premutagenic lesions. The aim of this thesis was to develop and validate a new tool for in vitro genotoxicity testing, based on the comet assay, that can be used in the elucidation of different mechanisms of action. This approach may represent a good candidate for complementing current in vitro genotoxicity testing batteries. The combination of the comet assay with lesion-specific enzymes is used to detect altered bases. A review about this version of the assay revealed that formamidopyrimidine DNA glycosylase (Fpg) is the most used enzyme, used for the detection of oxidized bases. In total, 12 different enzymes have been combined with the comet assay to detect other lesions such as alkylated bases, presence of uracil, pyrimidine dimers or AP-sites. The areas of application in which the enzyme-modified comet assay has been more extensively used are in vitro genotoxicity testing and human biomonitoring. For the detection of alkylated bases, two non-commercially available bacterial enzymes, 3-methyladenine DNA glycosylase II (AlkA) and 3-methyladenine DNA glycosylase (AlkD), have been sporadically employed. In this thesis, a commercial human alkyladenine DNA glycosilase (hAAG) was successfully applied for the first time. Moreover, the use of hAAG together with other different enzymes (non-commercial Fpg and commercial Fpg, Endonuclease III -Endo III- and human 8-oxoguanine DNA glycosylase -hOGG1-), with various specificities towards oxidized lesions, was optimized to be used on a single assay using a medium throughput format (i.e., 12 minigels/slide). To this aim, the incubation conditions when using the widely used 2 gels/slide format and the medium-throughput 12 minigels/slide format was assessed. This comparison highlighted that is crucial to perform enzyme titration experiments using the same protocol, equipment and the format that are going to be used in the final experiments. Moreover, in order to detect DNA cross-links, an extra DNA lesion which may be difficult to detect by mean of enzymes, an already known modification of the comet assay was set up using the same throughput format. Finally, both comet assay modifications were validated using TK-6 cells treated with non-cytotoxic concentrations of nine compounds with several mechanisms of action: oxidizing and alkylating agents, cross-linkers, a bulky-adducts inducer and non-genotoxic compounds. The combination of the results of both modifications allowed to clearly differentiate the induced lesions, with the exception of the bulky adducts, which was expected. Moreover, no DNA lesions was detected in cells treated with the non-genotoxic compounds. Both Fpg enzymes (non-commercial and commercial one) gave same results. The in vitro comet assay modified with several enzymes together with the cross-links modification increases significantly the comet assay ability to detect different premutagenic lesions, providing genotoxic mechanistic information about the type of damage. Its application might be very relevant in the current regulatory context

Efficacy and safety of tenecteplase in combination with enoxaparin, abciximab, or unfractionated heparin: The ASSENT-3 randomised trial in acute myocardial infarction

Background: Current fibrinolytic therapies fail to achieve optimum reperfusion in many patients. Low-molecular-weight heparins and platelet glycoprotein IIb/IIIa inhibitors have shown the potential to improve pharmacological reperfusion therapy. We did a randomised, open-label trial to compare the efficacy and safety of tenecteplase plus enoxaparin or abciximab, with that of tenecteplase plus weight-adjusted unfractionated heparin in patients with acute myocardial infarction. Methods: 6095 patients with acute myocardial infarction of less than 6 h were randomly assigned one of three regimens: full-dose tenecteplase and enoxaparin for a maximum of 7 days (enoxaparin group; n=2040), half-dose tenecteplase with weight-adjusted low-dose unfractionated heparin and a 12-h infusion of abciximab (abciximab group; n=2017), or full-dose tenecteplase with weight-adjusted unfractionated heparin for 48 h (unfractionated heparin group; n=2038). The primary endpoints were the composites of 30-day mortality, in-hospital reinfarction, or in-hospital refractory ischaemia (efficacy endpoint), and the above endpoint plus in-hospital intracranial haemorrhage or in-hospital major bleeding complications (efficacy plus safety endpoint). Analysis was by intention to treat. Findings: There were significantly fewer efficacy endpoints in the enoxaparin and abciximab groups than in the unfractionated heparin group: 233/2037 (11.4%) versus 315/2038 (15.4%; relative risk 0.74 [95% CI 0.63-0.87], p=0.0002) for enoxaparin, and 223/2017 (11.1%) versus 315/2038 (15.4%; 0.72 [0.61-0.84], p<0.0001) for abciximab. The same was true for the efficacy plus safety endpoint: 280/2037 (13.7%) versus 347/2036 (17.0%; 0.81 [0.70-0.93], p=0.0037) for enoxaparin, and 287/2016 (14.2%) versus 347/2036 (17.0%; 0.84 [0.72-0.96], p=0.01416) for abciximab. Interpretation: The tenecteplase plus enoxaparin or abciximab regimens studied here reduce the frequency of ischaemic complications of an acute myocardial infarction. In light of its ease of administration, tenecteplase plus enoxaparin seems to be an attractive alternative reperfusion regimen that warrants further study