Transferência gênica não viral para tendão : comparação de dois métodos

Background: tendons are part of the connective tissue that joins muscle to bone. Tendon injuries are a problem, since they have a poor ability to regenerate spontaneously. Alternative treatments involving the injection of local growth factors and gene transfer has been evaluated. Thus, we compared two methods for non-viral gene transfer tendons, using the GFP gene as reporter gene. Methods: Wistar rats had the medial quadriceps tendon exposed and the plasmid was transferred by direct injection or complexed with liposomes. Quantification of GFP in the tendom and in the spleen was evaluated by histological analysis with a fluorescence microscope. Results: gene transfer to the tendon was successfully obtained in both treatments. Lipoplex, as expected, showed the highest efficiency in transducing tenocytes, however we have found GFP expression also in the spleen. Naked DNA also showed fluorescence values above the control group and the signal was limited to the tendom. Discussion: the use of GFP as a reporter gene is a classical approach to evaluate gene transfer efficiency. Non-viral gene transfer methods are safe but show low levels of transduction and transient expression. For tendon repair, however, these characteristics may prove beneficial because a transient expression may be desirable to avoid the risk of adverse effects. GFP distribution in the spleen was probably a result of lipoplexes uptake by cells from the reticular endothelial system. Conclusion: taking into account the distribution of GFP in another tissue when using lipoplex, we believe that naked DNA is a more appropriate way to perform gene transfer to the tendon, ensuring safety, low cost and easy handling.Introdução: injúrias no tendão representam um problema, uma vez que estes têm pobre capacidade de regeneração espontânea. Tratamentos envolvendo injeção local de fatores de crescimento e transferência gênica tem sido avaliados. Assim, comparamos dois métodos de transferência gênica não viral para tendões, usando o gene GFP como gene repórter. Métodos: ratos Wistar tiveram a porção medial do tendão quadriciptal exposto e o plasmídeo foi transferido através de injeção direta ou complexado com lipossoma. A quantificação de GFP no tendão e no baço foi avaliada por análise histológica. Resultados: a transferência gênica para o tendão foi obtida com sucesso nos dois tratamentos. Lipoplexo demonstrou maior eficiência na transfecção, porém a presença de GFP foi detectada também no baço. A transfecção com DNA nu demonstrou valores de fluorescência superiores ao grupo controle e o sinal foi limitado ao tendão. Discussão: o uso de GFP como gene repórter é uma abordagem clássica para avaliar a eficiência da transferência de genes. A transferência não-viral é segura embora apresente expressão transiente. Para o reparo do tendão, no entanto, essas características podem ser benéficas, pois uma expressão transiente pode ser desejável para evitar o risco de efeitos adversos. A distribuição de GFP no baço foi provavelmente resultado da absorção dos lipoplexos por células do sistema retículo endotelial. Conclusão: twendo em conta a distribuição de GFP em outro tecido quando utilizamos lipoplexo, pensamos que o DNA nu é uma forma mais adequada para realizar a transferência de genes para o tendão, garantindo segurança, baixo custo e fácil manuseio

A simple architecture with self-assembled monolayers to build immunosensors for detecting the pancreatic cancer biomarker CA19-9

Accepted ManuscriptThe challenge of the early diagnosis of pancreatic cancer in routine clinical practice requires low-cost means of detection, and this may be achieved with immunosensors based on electrical or electrochemical principles. In this paper, we report a potentially low-cost immunosensor built with interdigitated gold electrodes coated with a self-assembled monolayer and a layer of anti-CA19-9 antibodies, which is capable of detecting the pancreatic cancer biomarker CA19-9 using electrical impedance spectroscopy. Due to specific, irreversible adsorption of CA19-9 onto its corresponding antibody, according to data from polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS), the immunosensor is highly sensitive and selective. It could detect CA19-9 in commercial samples with a limit of detection of 0.68 U mL−1, in addition to distinguishing between blood serum samples from patients with different concentrations of CA19-9. Furthermore, by treating the capacitance data with information visualization methods, we were able to verify the selectivity and robustness of the immunosensor with regard to false positives, as the samples containing higher CA19-9 concentrations, including those from tumor cells, could be distinguished from those with possible interferents.CAPES, FAPESP (Grant 2013/14262-7 and 2012/15543-7), CNPq (150985/2017-7), nBioNet network and Barretos Cancer Hospital for financial supportinfo:eu-repo/semantics/publishedVersio

Identification of hereditary cancer in the general population: development and validation of a screening questionnaire for obtaining the family history of cancer

One of the challenges for Latin American countries is to include in their healthcare systems technologies that can be applied to hereditary cancer detection and management. The aim of the study is to create and validate a questionnaire to identify individuals with possible risk for hereditary cancer predisposition syndromes (HCPS), using different strategies in a Cancer Prevention Service in Brazil. The primary screening questionnaire (PSQ) was developed to identify families at-risk for HCPS. The PSQ was validated using discrimination measures, and the reproducibility was estimated through kappa coefficient. Patients with at least one affirmative answer had the pedigree drawn using three alternative interview approaches: in-person, by telephone, or letter. Validation of these approaches was done. Kappa and intraclass correlation coefficients were used to analyze data’s reproducibility considering the presence of clinical criteria for HCPS. The PSQ was applied to a convenience sample of 20,000 women of which 3121 (15.6%) answered at least one affirmative question and 1938 had their pedigrees drawn. The PSQ showed sensitivity and specificity scores of 94.4% and 75%, respectively, and a kappa of 0.64. The strategies for pedigree drawing had reproducibility coefficients of 0.976 and 0.850 for the telephone and letter approaches, respectively. Pedigree analysis allowed us to identify 465 individuals (24.0%) fulfilling at least one clinical criterion for HCPS. The PSQ fulfills its function, allowing the identification of HCPS at-risk families. The use of alternative screening methods may reduce the number of excluded at-risk individuals/families who live in locations where oncogenetic services are not established.Research supported by Barretos Cancer Hospital. EIP has a grant from FAPESP (FAPESP, SP, Brazil, #2013/24633-2). N Campacci is supported by a PhD fellowship from FAPESP (FAPESP, SP, Brazil, #2015/02444-9).info:eu-repo/semantics/publishedVersio

Immunosensor for pancreatic cancer based on electrospun nanofibers coated with carbon nanotubes or gold nanoparticles

We report the fabrication of immunosensors based on nanostructured mats of electrospun nanofibers of polyamide 6 and poly(allylamine hydrochloride) coated either with multiwalled carbon nanotubes (MWCNTs) or gold nanoparticles (AuNPs), whose three-dimensional structure was suitable for the immobilization of anti-CA19-9 antibodies to detect the pancreatic cancer biomarker CA19-9. Using impedance spectroscopy, the sensing platform was able to detect CA19-9 with a detection limit of 1.84 and 1.57 U mL-1 for the nanostructured architectures containing MWCNTs and AuNPs, respectively. The high sensitivity achieved can be attributed to the irreversible adsorption between antibodies and antigens, as confirmed with polarization-modulated infrared reflection absorption spectroscopy. The adsorption mechanism was typical Langmuir-Freundlich processes. The high sensitivity and selectivity of the immunosensors were also explored in tests with blood serum from patients with distinct concentrations of CA19-9, for which the impedance spectra data were processed with a multidimensional projection technique. The robustness of the immunosensors in dealing with patient samples without suffering interference from analytes present in biological fluids is promising for a simple, effective diagnosis of pancreatic cancer at early stages.This work was supported by FAPESP (Grant 2013/14262-7), CNPq, and CAPES (Brazil). D.S.C. also thanks FAPESP (Grant 2014/16789-5), Embrapa−Rede Agronano, and MCTI SisNano for financial support.info:eu-repo/semantics/publishedVersio

The Barretos Cancer Hospital Animal Facility: implementation and results of a dedicated platform for preclinical oncology models

The Barretos Cancer Hospital Animal Facility (BCHAF) is a unique facility in Brazil exclusively dedicated to working with animal models for cancer research. In this article, we briefly present our modern facility and the main experiments performed, focusing on mutant strains of mice (PTCH-knockout and ApcMin mice), xenograft models, and patient-derived xenografts (PDXs). Our results show the progress and challenges in establishing these models and the need for having an appropriate representation of our cancer population to better understand tumor biology and to identify cancer biomarkers, which could be putatively targeted, allowing for personalized therapy.This study was funded by the Public Ministry of Labor Campinas (Research, Prevention and Education of Occupational Cancer) and by Pio XII Foundation, Barretos Cancer Hospital internal funds, Grant Number: 13/2021

Terapia celular autóloga para lesões osteo-condrais : estudo em um modelo animal em coelhos

Resumo não disponível

Transferência gênica não viral para tendão : comparação de dois métodos

Background: tendons are part of the connective tissue that joins muscle to bone. Tendon injuries are a problem, since they have a poor ability to regenerate spontaneously. Alternative treatments involving the injection of local growth factors and gene transfer has been evaluated. Thus, we compared two methods for non-viral gene transfer tendons, using the GFP gene as reporter gene. Methods: Wistar rats had the medial quadriceps tendon exposed and the plasmid was transferred by direct injection or complexed with liposomes. Quantification of GFP in the tendom and in the spleen was evaluated by histological analysis with a fluorescence microscope. Results: gene transfer to the tendon was successfully obtained in both treatments. Lipoplex, as expected, showed the highest efficiency in transducing tenocytes, however we have found GFP expression also in the spleen. Naked DNA also showed fluorescence values above the control group and the signal was limited to the tendom. Discussion: the use of GFP as a reporter gene is a classical approach to evaluate gene transfer efficiency. Non-viral gene transfer methods are safe but show low levels of transduction and transient expression. For tendon repair, however, these characteristics may prove beneficial because a transient expression may be desirable to avoid the risk of adverse effects. GFP distribution in the spleen was probably a result of lipoplexes uptake by cells from the reticular endothelial system. Conclusion: taking into account the distribution of GFP in another tissue when using lipoplex, we believe that naked DNA is a more appropriate way to perform gene transfer to the tendon, ensuring safety, low cost and easy handling.Introdução: injúrias no tendão representam um problema, uma vez que estes têm pobre capacidade de regeneração espontânea. Tratamentos envolvendo injeção local de fatores de crescimento e transferência gênica tem sido avaliados. Assim, comparamos dois métodos de transferência gênica não viral para tendões, usando o gene GFP como gene repórter. Métodos: ratos Wistar tiveram a porção medial do tendão quadriciptal exposto e o plasmídeo foi transferido através de injeção direta ou complexado com lipossoma. A quantificação de GFP no tendão e no baço foi avaliada por análise histológica. Resultados: a transferência gênica para o tendão foi obtida com sucesso nos dois tratamentos. Lipoplexo demonstrou maior eficiência na transfecção, porém a presença de GFP foi detectada também no baço. A transfecção com DNA nu demonstrou valores de fluorescência superiores ao grupo controle e o sinal foi limitado ao tendão. Discussão: o uso de GFP como gene repórter é uma abordagem clássica para avaliar a eficiência da transferência de genes. A transferência não-viral é segura embora apresente expressão transiente. Para o reparo do tendão, no entanto, essas características podem ser benéficas, pois uma expressão transiente pode ser desejável para evitar o risco de efeitos adversos. A distribuição de GFP no baço foi provavelmente resultado da absorção dos lipoplexos por células do sistema retículo endotelial. Conclusão: twendo em conta a distribuição de GFP em outro tecido quando utilizamos lipoplexo, pensamos que o DNA nu é uma forma mais adequada para realizar a transferência de genes para o tendão, garantindo segurança, baixo custo e fácil manuseio

Prevalence of the serpin peptidase inhibitor (alpha-1-antitrypsin) PI*S and PI*Z alleles in Brazilian children with liver disease

Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 (SERPINA1) deficiency is one of the main genetic causes related to liver disease in children. In SERPINA1 deficiency the most frequent SERPINA1 alleles found are the PI*S and PI*Z alleles. We used the polymerase chain reaction and the amplification created restriction site (ACRS) technique to investigate the prevalence of the PI*S and PI*Z alleles in a group of Brazilian children (n = 200) with liver disease and established the general frequency of the PI*S allele in our population. We found a significant association of the PI*Z allele and liver disease, but no such relationship was found for the PI*S allele. Our results show that SERPINA1 deficiency due to the PI*Z allele, even when heterozygous, is a frequent cause of liver disease in our group of Brazilian children but that the PI*S allele does not confer an increased risk of hepatic disorders in our group of Brazilian childre