Biodentine Induce Proteínas Apoptóticas Caspasa 3 y PARP-1 en Células de la Pulpa Dental Humana / Biodentine Induces Apoptotic Proteins Caspase 3 and PARP - 1 on Human Dental Pulp Cells

RESUMEN: Las patologías pulpares han sido un verdadero reto para la odontología principalmente por su tratamiento. Actualmente, existen numerosos biomateriales en el mercado que reportan tener propiedades inherentes en los tejidos dentarios. Sin embargo, diferentes estudios sobre múltiples líneas celulares expuestas a estos biomateriales demuestran resultados controversiales como biocompatiblidad y citotoxicidad celular. Biodentine, es un cemento endodóntico en base a silicatos cálcico de múltiples aplicaciones, que prestaría propiedades de biocompatibilidad como bioactividad celular, características que le permitirían incluso ser utilizado en contacto directo con la pulpa dental. El objetivo de este estudio es la evaluación in-vitro de Biodentine, sobre cultivos de células de la pulpa dental humana (CCPDH). Se prepararon discos de cemento de Biodentine™ de 2 x 6 mm, los que se expusieron a cultivos de células aisladas de la pulpa dental humana. Luego de 24, 48 y 72 horas de exposición, se realizaron ensayos de viabilidad celular utilizando el método colorimétrico MTT. También se realizaron ensayos de expresión proteica de dos proteínas involucradas en la vía de señalización de la apoptosis celular: Caspasa - 3 clivada y Poli (ADP-Ribosa) Polimerasa, PARP - 1. Existen diferencias estadísticamente significativas (p<0,05) en los ensayos de viabilidad celular entre las células expuestas a Biodentine y el grupo control, como también a medida que aumenta el tiempo de exposición (p<0,05). Por otra parte, también existen diferencias significativas (p<0,05) en la expresión de PARP- 1 en los grupos sometidos a Biodentine. Los resultados obtenidos en este estudio demuestran que Biodentine genera citotoxicidad celular en cultivos celulares de pulpa dental humana, por disminución de la viabilidad celular como por la expresión de proteínas apoptóticas. Es por esto que la utilización de este biomaterial debería ser estudiado y considerarse en cada caso clínico, especialmente como recubridor pulpar directo.

ABSTRACT: Oral pathologies have been a real challenge for dentistry, mainly due to its treatment. Currently, there are numerous biomaterials on the market that may present inherent properties in dental tissues. However, studies on multiple cell lines are based on biocompatible results such as biocompatibility and cellular cytotoxicity. Biodentine is endodontic cement based on calcium silicates of multiple applications, which would provide biocompatibility properties as cellular bioactivity, characteristics that will allow it to be used in direct contact with the dental pulp. The objective of this study is the in vitro evaluation of Biodentine, on cultures of cells of the human dental pulp (HDPC). Biodentine cement disks of 2 x 6 mm were prepared, and HDPC culture plates were introduced. After 24, 48 and 72 hours of exposure, cell viability tests were performed using the MTT colorimetric method. On the other hand, protein expression assays of two proteins involved in the signaling pathway of cell apoptosis Caspase-3 cleaved (cas-3 clv) and PARP-1 are carried out. There are statistically significant differences (p <0,05) in the cell viability tests between Biodentine and control group, as well as the exposure time increases (p <0,05). Otherwise, there are also significant differences (p <0,05) in the expression of PARP-1 in the groups, sometimes a Biodentine. The results in this study that Biodentine generates a cellular cytotoxicity in HDPC cultures, therefore, cell viability as the expression of apoptotic proteins. This is why the use of this biomaterial should be studied for each particular clinical case, especially as a direct pulp capping agent.

PLAG1, SOX10, and Myb Expression in Benign and Malignant Salivary Gland Neoplasms

BACKGROUND: Recent findings in molecular pathology suggest that genetic translocation and/or overexpression of oncoproteins is important in salivary gland tumorigenesis and diagnosis. We investigated PLAG1, SOX10, and Myb protein expression in various salivary gland neoplasm tissues. METHODS: A total of 113 cases of surgically resected salivary gland neoplasms at the National Cancer Center from January 2007 to March 2017 were identified. Immunohistochemical staining of PLAG1, SOX10, and Myb in tissue samples was performed using tissue microarrays. RESULTS: Among the 113 cases, 82 (72.6%) were benign and 31 (27.4%) were malignant. PLAG1 showed nuclear staining and normal parotid gland was not stained. Among 48 cases of pleomorphic adenoma, 29 (60.4%) were positive for PLAG1. All other benign and malignant salivary gland neoplasms were PLAG1-negative. SOX10 showed nuclear staining. In normal salivary gland tissues SOX10 was expressed in cells of acinus and intercalated ducts. In benign tumors, SOX10 expression was observed in all pleomorphic adenoma (48/48), and basal cell adenoma (3/3), but not in other benign tumors. SOX10 positivity was observed in nine of 31 (29.0%) malignant tumors. Myb showed nuclear staining but was not detected in normal parotid glands. Four of 31 (12.9%) malignant tumors showed Myb positivity: three adenoid cystic carcinomas (AdCC) and one myoepithelial carcinoma with focal AdCC-like histology. CONCLUSIONS: PLAG1 expression is specific to pleomorphic adenoma. SOX10 expression is helpful to rule out excretory duct origin tumor, but its diagnostic value is relatively low. Myb is useful for diagnosing AdCC when histology is unclear in the surgical specimen.

Effect of DSA on Vascular Endothelial Cell Injury in NK Cell -Mediated ADCC / 中国实验血液学杂志

OBJECTIVE@#To investigate the injury effect of anti-donor specific antibody (DSA) on human umbilical vein enolothelial cells (HUVEC) in NK-mediated antibody-dependent cell cytotoxity (ADCC).@*METHODS@#The peripheral blood of 10 healthy donors was colleced for allo-HSCT of AML patients diagnosed in Department of Hemology of the Tumor Hospital affiliated to Shanxi Medical University, then the peripheral blood NK cells were isolated and used as the effector cells; the HUVEC of passages 9-6 were selected and co-cultured with DSA, then the DSA-binding HUVEC were used as the target cells (CDH group), while the DSA-unbinding HUVEC were used as negative control (UDH group). After co-culture of effecor cells with target cells, the expression of IFN-γ was detected by flow cytometry and the HUVEC activity was detected by using MTT method, so as to indirectily reflect the injury effect of DSA-mediated ADCC on endothelial cells.@*RESULTS@#With the increase of effector-target (E:T) ratio, the activity of HUVEC decreased, the expression level of IFN-γ increased. Under the some effector-target ratio (1∶1, 10∶1, 20∶1), the activity of HUVEC in CDH group was significantly lower than that of UDH group, and the expression of IFN-γ was significantly higher than that of the UDH group (P<0.05).@*CONCLUSION@#DSA can damage vascular endothelial cells through the ADCC effect mediated by NK cells.

Donor Specific Antibody Negative Antibody-Mediated Rejection after ABO Incompatible Liver Transplantation / 대한이식학회지

Antibody-mediated rejection (AMR) is a major complication after ABO-incompatible liver transplantation. According to the 2016 Banff Working Group on Liver Allograft Criteria for the diagnosis of acute AMR, a positive serum donor specific antibody (DSA) is needed. On the other hand, the clinical significance of the histological findings of AMR in the absence of DSA is unclear. This paper describes a 57-year-old man (blood type, O+) who suffered from hepatitis B virus cirrhosis with hepatocellular carcinoma. Pre-operative DSA and cross-matching were negative. After transplantation, despite the improvement of the liver function, acute AMR was observed in the protocol biopsy on postoperative day 7; the cluster of differentiation 19+ (CD19+) count was 0% and anti-ABO antibody titers were 1:2. This paper presents the allograft injury like AMR in the absence of DSA after ABOi living donor liver transplantation with low titers of anti-ABO antibody and depleted serum CD19+ B cells.

Optimization of Large-Scale Expansion and Cryopreservation of Human Natural Killer Cells for Anti-Tumor Therapy

Allogeneic natural killer (NK) cell therapy is a potential therapeutic approach for a variety of solid tumors. We established an expansion method for large-scale production of highly purified and functionally active NK cells, as well as a freezing medium for the expanded NK cells. In the present study, we assessed the effect of cryopreservation on the expanded NK cells in regards to viability, phenotype, and anti-tumor activity. NK cells were enormously expanded (about 15,000-fold expansion) with high viability and purity by stimulating CD³⁺ T cell-depleted peripheral blood mononuclear cells (PBMCs) with irradiated autologous PBMCs in the presence of IL-2 and OKT3 for 3 weeks. Cell viability was slightly reduced after freezing and thawing, but cytotoxicity and cytokine secretion were not significantly different. In a xenograft mouse model of hepatocellular carcinoma cells, cryopreserved NK cells had slightly lower anti-tumor efficacy than freshly expanded NK cells, but this was overcome by a 2-fold increased dose of cryopreserved NK cells. In vivo antibody-dependent cell cytotoxicity (ADCC) activity of cryopreserved NK cells was also demonstrated in a SCID mouse model injected with Raji cells with rituximab co-administration. Therefore, we demonstrated that expanded/frozen NK cells maintain viability, phenotype, and anti-tumor activity immediately after thawing, indicating that expanded/frozen NK cells can provide ‘ready-to-use’ cell therapy for cancer patients.

Engineering of Anti-CD133 Trispecific Molecule Capable of Inducing NK Expansion and Driving Antibody-Dependent Cell-Mediated Cytotoxicity / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The selective elimination of cancer stem cells (CSCs) in tumor patients is a crucial goal because CSCs cause drug refractory relapse. To improve the current conventional bispecific immune-engager platform, a 16133 bispecific natural killer (NK) cell engager (BiKE), consisting of scFvs binding FcγRIII (CD16) on NK cells and CD133 on carcinoma cells, was first synthesized and a modified interleukin (IL)-15 crosslinker capable of stimulating NK effector cells was introduced. MATERIALS AND METHODS: DNA shuffling and ligation techniques were used to assemble and synthesize the 1615133 trispecific NK cell engager (TriKE). The construct was tested for its specificity using flow cytometry, cytotoxic determinations using chromium release assays, and lytic degranulation. IL-15–mediated expansion was measured using flow-based proliferation assays. The level of interferon (IFN)-γ release was measured because of its importance in the anti-cancer response. RESULTS: 1615133 TriKE induced NK cell–mediated cytotoxicity and NK expansion far greater than that achieved with BiKE devoid of IL-15. The drug binding and induction of cytotoxic degranulation was CD133+ specific and the anti-cancer activity was improved by integrating the IL-15 cross linker. The NK cell–related cytokine release measured by IFN-γ detection was higher than that of BiKE. NK cytokine release studies showed that although the IFN-γ levels were elevated, they did not approach the levels achieved with IL-12/IL-18, indicating that release was not at the supraphysiologic level. CONCLUSION: 1615133 TriKE enhances the NK cell anti-cancer activity and provides a self-sustaining mechanism via IL-15 signaling. By improving the NK cell performance, the new TriKE represents a highly active drug against drug refractory relapse mediated by CSCs.

Anti-GD2 Monoclonal Antibody (dinutuximab) with GM-CSF, Interleukin 2, and Cis-retinoic Acid for the Treatment of High-risk Neuroblastoma / 임상소아혈액종양

Neuroblastoma, one of the most common solid tumors in early childhood, exhibits aberrant cell-surface glycosylation patterns. In neuroblastoma, disialoganglioside (GD2) is expressed homogeneously and abundantly on 100% of neuroblastoma cells. GD2 is a good tumor marker for developing an anti-tumor-monoclonal antibody (mAb) to neuroblastoma. Immunotherapy, using anti-GD2-mAb, has been tried since last 20 years to improve the prognosis of high risk neuroblastoma patients who show a 5-year survival rate of less than 30% regardless of an intense multimodal therapy. Since the first clinical trial of murine anti-GD2-mAb 3F8 had been performed, multiple clinical studies showed that anti-GD2-mAb might improve the prognosis of high risk neuroblastoma patients. Anti-GD2-mAb removes the neuroblastoma cells via apoptosis by antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity. To elicit a stronger ADCC response to antibody therapy, cytokines such as, GM-CSF and interleukin-2 are concomitantly administered, which stimulate the natural anti-tumor activity of the immune system. Children's Oncology Group performed a study of chimeric anti-GD2-mAb (ch14.18) administration with GM-CSF, IL-2 for high risk neuroblastoma patients and showed the improvement of overall survival rate. Based on this study US FDA approved the chimeric anti-GD2-mAb (commercially manufactured dinutuximab) for the treatment of high risk neuroblastoma. Dinutuximab is the the first mAb for use in combination of cytokines for the maintenance treatment of pediatric patients with high risk neuroblastoma who achieve at least a partial response to intensified multimodal therapy. The first anti-tumor-mAb used for children, dinutuximab, could be the base of further development of mAb against the cancers in childhood.

Anti-GD2 Monoclonal Antibody (dinutuximab) with GM-CSF, Interleukin 2, and Cis-retinoic Acid for the Treatment of High-risk Neuroblastoma / 임상소아혈액종양

Neuroblastoma, one of the most common solid tumors in early childhood, exhibits aberrant cell-surface glycosylation patterns. In neuroblastoma, disialoganglioside (GD2) is expressed homogeneously and abundantly on 100% of neuroblastoma cells. GD2 is a good tumor marker for developing an anti-tumor-monoclonal antibody (mAb) to neuroblastoma. Immunotherapy, using anti-GD2-mAb, has been tried since last 20 years to improve the prognosis of high risk neuroblastoma patients who show a 5-year survival rate of less than 30% regardless of an intense multimodal therapy. Since the first clinical trial of murine anti-GD2-mAb 3F8 had been performed, multiple clinical studies showed that anti-GD2-mAb might improve the prognosis of high risk neuroblastoma patients. Anti-GD2-mAb removes the neuroblastoma cells via apoptosis by antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity. To elicit a stronger ADCC response to antibody therapy, cytokines such as, GM-CSF and interleukin-2 are concomitantly administered, which stimulate the natural anti-tumor activity of the immune system. Children's Oncology Group performed a study of chimeric anti-GD2-mAb (ch14.18) administration with GM-CSF, IL-2 for high risk neuroblastoma patients and showed the improvement of overall survival rate. Based on this study US FDA approved the chimeric anti-GD2-mAb (commercially manufactured dinutuximab) for the treatment of high risk neuroblastoma. Dinutuximab is the the first mAb for use in combination of cytokines for the maintenance treatment of pediatric patients with high risk neuroblastoma who achieve at least a partial response to intensified multimodal therapy. The first anti-tumor-mAb used for children, dinutuximab, could be the base of further development of mAb against the cancers in childhood.

Efeitos do ácido fólico não metabolizado na metilação global do DNA, na expressão de RNAm dos genes de DHFR, MTHFR, interferon-γ, TNF-α e interleucina-8, e na citotoxicidade das células NK / Effects of unmetabolized folic acid on global DNA methylation, on mRNA expression of DHFR, MTHFR, interferon-α, TNF-α and interleukin-8 genes, and on NK cells cytotoxicity

Com o início da fortificação de farinhas com ácido fólico (AF) no Brasil, a partir 2004, a população passou a estar exposta de modo compulsório a maiores quantidades desta vitamina. Sabe-se que o AF na sua forma sintética pode não ser completamente convertido para formas metabolicamente ativas, levando ao aparecimento de uma fração não metabolizada no organismo. Este fato é mais preocupante nos indivíduos que além da fortificação, fazem uso terapêutico dessa vitamina, como em pacientes com anemias hemolíticas (esferocitose hereditária (EH), ß-talassemia heterozigótica (ß-TH), entre outras). O objetivo desse estudo foi avaliar se as concentrações séricas de AF não metabolizado (UMFA) afetam a metilação global do DNA; a expressão de RNAm de genes da DHFR, MTHFR, interferon-γ, TNF-α e interleucina (IL)-8; e a citotoxicidade de células NK. Foram incluídos 27 pacientes com EH, 50 indivíduos com ß-talassemia heterozigótica (ß-TH) e 136 indivíduos saudáveis. Outros 30 indivíduos saudáveis foram incluídos num estudo de intervenção com 5mg/dia de AF durante 90 dias. Foram realizadas as análises de: ácido fólico sérico, eritrocitário e UMFA, vitamina B12, homocisteína total; expressões de RNAm dos genes de MTHFR, DHFR, IFN-γ, TNF-α e IL-8; citotoxicidade das células NK; metilação global do DNA e citocinas séricas IL6, IL-8, IL10, IFN-γ e TNF-α. Resultados: Os pacientes EH apresentaram maiores concentrações de AF (sérico, eritrocitário e UMFA) que seus controles, sendo que 55,5% (método microbiológico) e 74,1% (método LC-MS/MS) apresentaram concentrações suprafisiológicas da vitamina, e 74,1% apresentaram concentrações aumentadas de UMFA. No grupo ß-TH foi observado maiores concentrações de folato eritrocitário comparado ao controle e 22% dos indivíduos tinham concentrações suprafisiológicas de AF. No estudo de intervenção, após 45 e 90 dias de uso de AF as concentrações suprafisiológicas estavam presentes em 93,3% dos indivíduos e 100% deles apresentavam concentrações aumentadas de UMFA. Não foram observadas diferenças nas taxas de metilação global do DNA entre os grupos EH e ß-TH quando comparados aos seus controles e não foi verificada correlação entre metilação global e concentrações de UMFA. Tanto EH quanto ß-TH apresentaram maior expressão de RNAm de IL-8, quando comparados aos seus controles. No grupo de intervenção houve maior expressão de IL-8 após 45 dias de uso de AF quando comparado ao período pré-intervenção. Foi mostrada uma correlação inversa entre as concentrações de folato eritrocitário com o número de células NK no grupo EH e com a capacidade citotóxica das células NK no grupo total (EH + controle). No grupo intervenção foi observado menor número e menor capacidade citotóxica das células NK após 90 dias de uso de AF. Conclusões: O uso de AF 5mg/dia foi associado com aumento expressivo das concentrações de folato sérico, eritrocitário, UMFA, na expressão de RNAm de genes da citocina inflamatória IL-8 e redução do número e da citotoxicidade das células NK. Dessa forma, altas doses de AF podem resultar em alterações de componentes do sistema imune podendo prejudicar os mecanismos de vigilância celular das células NK. Os nossos achados sugerem que é importante o acompanhamento terapêutico dos pacientes que fazem uso de AF, especialmente aqueles indivíduos que fazem uso crônico desta vitamina por longo tempo, como os pacientes com anemias hemolíticas, mulheres que desejam engravidar e gestantes

With the beginning of the fortification of flour with folic acid (FA) in Brazil, since 2004, the population has been exposed compulsorily to larger amounts of this vitamin. It is known that FA in its synthetic form can not be completely converted to metabolically active forms, leading to the appearance of an unmetabolized fraction in the body. This fact is more worrying in subjects beyond fortification, make therapeutic use of this vitamin, such as patients with hemolytic anemia (hereditary spherocytosis (HS), ß-thalassemia heterozygotic (ß-TH), among others). The aim of this study was to evaluate if serum concentrations of unmetabolized FA (UMFA) affect the global DNA methylation; mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin (IL)-8 genes; and on cytotoxicity of NK cells. It was included 27 patients EH, 50 subjects ß-TH and 136 healthy subjects. Another 30 healthy subjects were included in an intervention study with 5 mg/FA daily during 90 days. Analyzes performed were: serum and erythrocyte folate, and UMFA, vitamin B12, tHcy; mRNA expression of MTHFR, DHFR, IFN-γ, TNF-α and IL-8 genes; cytotoxicity of NK cells; global DNA methylation and serum cytokines IL6, IL-8, IL10, IFN-γ and TNF-α. Results: EH patients presented higher FA concentrations (serum, erythrocytes and UMFA) than controls ones, and 55.5% (microbiologic method) and 74.1% (LC-MS/MS method) showed supraphysiologic concentrations of vitamin, and 74.1% presented increased concentrations of UMFA. In ß-TH group, it was observed higher erythrocyte folate concentrations compared with the control and 22% of subjects had supraphysiological concentrations of FA. In the intervention study, after 45 and 90 days of FA use, supraphysiological concentrations were present in 93.3% of subjects and 100% of them showed increased concentrations of UMFA. It was not observed differences in global methylation of DNA between EH and ß-TH groups when compared to their controls and it was not observed significant correlation between global DNA methylation and UMFA levels. Both EH and ß-TH showed higher mRNA expression of IL-8 gene, when compared to controls. In the intervention group, there was higher mRNA expression of IL-8 gene after 45 days of FA use when compared to pre-intervention period. It was demonstrated an inverse correlation between erythrocyte folate levels and the number of NK cells in EH group; and cytotoxic capacity of NK cells on total group (EH + control). In intervention group, it was observed fewer number and lower cytotoxic capacity of NK cells after 90 days of AF use. Conclusions: The use of AF 5mg daily was associated with a significant increase in serum and erythrocytes folate levels, accompanied by increase in UMFA levels, an increase in mRNA expression of IL-8 gene, and reduction of the number and the cytotoxicity capacity of NK cells. Thus, high doses of AF can result in modifications of the immune system components, which may damage the cell surveillance mechanisms of NK cells. Our findings suggest that is important the therapeutic follow up of patients that are using AF, especially those subjects with chronic use of this vitamin, such as patients with hemolytic anemia, women who wish to become pregnant and pregnant women

Effect of FCGR3A Polymorphisms on Antibody-dependent Cetuximab-mediated Cytotoxicity in A549 Cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the effect of FCGR3A polymorphisms on the antibody-dependent cell-mediated cytotoxicity (ADCC) activity induced by cetuximab against A549 cells.</p><p><b>METHODS</b>A549 cell line was used as target cells and NKTm cells as effector cells. FCGR3A polymorphisms were detected by direct sequencing. The ADCC activity mediated by cetuximab was assessed by CCK-8 assay.</p><p><b>RESULTS</b>Three genotypes of FCGR3A were detected:V/V,V/F,and F/F. The ADCC activity of NKTm cells with these three different genotypes mediated by cetuximab were significantly different (P=0.0015). NKTm cells with FCGR3A-158V/V genotypes had significantly higher ADCC activity than FCGR3A-V/F or F/F genotypes (P<0.01),whereas the ADCC activity between V/F and F/F genotype showed no statistical significance(P>0.05).</p><p><b>CONCLUSION</b>FCGR3A polymorphisms have an impact on ADCC activity mediated by cetuximab in NKTm cells.</p>

Nutritional repercussions in patients submitted to bariatric surgery / Repercussões nutricionais em pacientes submetidos à cirurgia bariátrica

BACKGROUND: Few studies evaluated the association between nutritional disorders, quality of life and weight loss in patients undergoing bariatric surgery. AIM: To identify nutritional changes in patients undergoing bariatric surgery and correlate them with weight loss, control of comorbidities and quality of life. METHOD: A prospective cohort, analytical and descriptive study involving 59 patients undergoing bariatric surgery was done. Data were collected preoperatively at three and six months postoperatively, evaluating nutritional aspects and outcomes using BAROS questionnaire. The data had a confidence interval of 95%. RESULTS: The majority of patients was composed of women, 47 (79.7%), with 55.9% of the series with BMI between 40 to 49.9 kg/m². In the sixth month after surgery scores of quality of life were significantly higher than preoperatively (p<0.05) and 27 (67.5 %) patients had comorbidities resolved, 48 (81.3 %) presented BAROS scores of very good or excellent. After three and six months of surgery 16 and 23 presented some nutritional disorder, respectively. There was no relationship between the loss of excess weight and quality of life among patients with or without nutritional disorders. CONCLUSION: Nutritional disorders are uncommon in the early postoperative period and, when present, have little or no influence on quality of life and loss of excess weight. .

RACIONAL: Poucos estudos avaliam a associação entre distúrbios nutricionais, qualidade de vida e perda de peso em pacientes submetidos à cirurgia bariátrica. OBJETIVO: Identificar alterações nutricionais em pacientes submetidos à cirurgia bariátrica e correlacioná-las com perda de peso, controle de comorbidades e qualidade de vida. MÉTODO: Estudo de coorte, prospectivo, analítico e descritivo envolvendo 59 pacientes submetidos à cirurgia bariátrica. Os dados foram coletados no pré-operatório e aos três e seis meses pós- operatórios, quantificando aspectos nutricionais e utilizando o Bariatric Analysis and Reporting Outcomes System (BAROS) como ferramenta de sucesso. Os dados usaram intervalo de confiança de 95%. RESULTADOS: O total de mulheres foi 47 (79,7%), sendo 55,9% com IMC entre 40-49,9 kg/m². No sexto mês depois da operação os escores de qualidade de vida foram significativamente maiores do que no pré-operatório (p<0,05) e 27 (67,5%) pacientes tinham todas comorbidades resolvidas, 48 (81,3%) apresentaram conceito BAROS muito bom ou excelente. Após três e seis meses 16 e 23 pacientes apresentaram algum distúrbio nutricional, respectivamente. Não houve relação entre a perda do excesso de peso e qualidade de vida entre pacientes com ou sem distúrbio nutricional. CONCLUSÃO: os distúrbios nutricionais são pouco frequentes no pós-operatório precoce e, quando presentes, têm pouca ou nenhuma influência na qualidade de vida e na perda do excesso de peso. .

Development of a novel reporter gene method for determination of ADCC potency of anti-CD20 monoclonal antibody / 药学学报

The biological activity of ADCC by anti-CD20 monoclonal antibody was determined by BioGlo™ Luciferase Assay System using Jurkat/NFAT-luc+FcγRIIIa cell line as effector cell and WIL2-S cell line as target cell. The developed method was verified for specificity, precision and accuracy. Anti-CD20 monoclonal antibody showed a dose-response mode by the developed method, and the determination result complied with the following four-parameter equation: y = (A-D)/[1 + (X/C)(B)] + D. The optimized parameters of the method were determined including the antibodies diluted concentration (18,000 ng·mL(-1)), dilution rate (1:5), the ratio of effector cell and target cell (6:1), and induction time (6 h). The values of eight independent tests have passed a statistical test for curve regression analysis, linear or parallelism, which showed the method possessed good specificity. Four different dilute groups of recovery rates sample were determined for 3 times, and the result showed mean relative potencies of (44.39±3.93)%, (72.74±2.78)%, (128.28±7.01)% and (168.19±2.70)% respectively, with a variation coefficient of less than 10%, and the recoveries of (88.78±7.85)%, (96.99±3.70)%, (102.63±5.61)% and (112.12±1.80)% respectively. A novel reporter gene method for determination of biological activity of ADCC by anti-CD20 monoclonal antibody was successfully developed, which showed strong specificity, good reproducibility and high accuracy, and might be used routinely.

Comprehensive Cytomorphologic Analysis of Pulmonary Adenoid Cystic Carcinoma: Comparison to Small Cell Carcinoma and Non-pulmonary Adenoid Cystic Carcinoma

BACKGROUND: Cytologic diagnosis of pulmonary adenoid cystic carcinoma (AdCC) is frequently challenging and differential diagnosis with small cell carcinoma is often difficult. METHODS: Eleven cytologically diagnosed cases of pulmonary AdCC were collected and reviewed according to fifteen cytomorphologic characteristics: small cell size, cellular uniformity, coarse chromatin, hyperchromasia, distinct nucleolus, frequent nuclear molding, granular cytoplasm, organoid cluster, sheet formation, irregular border of cluster, hyaline globule, hyaline basement membrane material, individual cell necrosis or apoptotic body, and necrotic background. Twenty cases of small cell carcinoma and fifteen cases of non-pulmonary AdCC were also reviewed for the comparison. RESULTS: Statistically significant differences were identified between pulmonary AdCC and small cell carcinoma in fourteen of the fifteen cytomorphologic criteria (differences in sheet formation were not statistically significant). Cellular uniformity, distinct nucleolus, granular cytoplasm, distinct cell border, organoid cluster, hyaline globule, and hyaline basement membrane material were characteristic features of AdCC. Frequent nuclear molding, individual cell necrosis, and necrotic background were almost exclusively identified in small cell carcinoma. Although coarse chromatin and irregular cluster border were observed in both, they favored the diagnosis of small cell carcinoma. Hyaline globules were more frequently seen in non-pulmonary AdCC cases. CONCLUSIONS: Using the fifteen cytomorphologic criteria described by this study, pulmonary AdCC could be successfully distinguished from small cell carcinoma. Such a comprehensive approach to an individual case is recommended for the cytologic diagnosis of pulmonary AdCC.

Evaluation of biological activities of Physalis peruviana ethanol extracts and expression of Bcl-2 genes in HeLa cells

Physalis species are used in folk medicine for phytotherapeutic properties. The extracts of medicinal plants are known to possesscytotoxic and chemopreventative compounds. In this study we investigated antibacterial, antioxidant, DNA damage preventativeproperties of Physalis peruviana (golden berry) on leaf and shoot ethanol extracts and their effects on cytotoxicity of HeLa cellsand expression of apoptotic pathway genes. Among the tested bacteria for antibacterial activity, maximum inhibition zone wasdetermined in Lactococcus lactis. The phenolic content was found higher in leaf extracts than shoot extracts. The antioxidantactivity showed the highest TEAC values of the leaf (2 mg/mL) and the shoot (0.5 mg/mL) extracts as 0.291±0.04 and 0.192±0.015,respectively. In DNA damage prevention assay both leaf and shoot extracts, especially 30 and 20 µg/mL concentrations, exhibitedsignificant protection against DNA damage-induced by hydroxyl radical generated by Fenton reaction. Our results suggest thatleaf and shoot extracts possess cytotoxic effect on HeLa cells when applied as 100 µg/mL concentration. Also mRNA expressionanalysis showed the alteration of antiapoptotic genes, so the results suggest that P. peruviana ethanol extracts induce apoptoticcell death and should be investigated for identification of active compounds and their mechanisms of action.

Cytotoxicity and DNA damage in the neutrophils of patients with sickle cell anaemia treated with hydroxyurea

Hydroxyurea (HU) is the most important advance in the treatment of sickle cell anaemia (SCA) for preventing complications and improving quality of life for patients. However, some aspects of treatment with HU remain unclear, including their effect on and potential toxicity to other blood cells such as neutrophils. This study used the measurement of Lactate Dehydrogenase (LDH) and Methyl ThiazolTetrazolium (MTT) and the comet assay to investigate the cytotoxicity and damage index (DI) of the DNA in the neutrophils of patients with SCA using HU.In the LDH and MTT assays, a cytoprotective effect was observed in the group of patients treated, as well as an absence of toxicity. When compared to patients without the treatment, the SS group (n=20, 13 women and 07 men, aged 18-69 years), and the group of healthy individuals (AA) used as a control group (n=52, 28 women and 24 men, aged 19-60 years), The SSHU group (n=21, 11 women and 10 men, aged 19-63 years) showed a significant reduction (p<0.001) in LDH activity and an increase in the percentage of viable cells by the MTT (p<0.001). However, the SSHU group presented significantly higher DI values (49.57±6.0 U/A) when compared to the AA group (7.43 ± 0,94U/A) and the SS group (22.73 ±5.58 U/A) (p<0.0001), especially when treated for longer periods (>20 months), demonstrating that despite the cytoprotective effects in terms of cell viability, the use of HU can induce DNA damage in neutrophils.

A hidroxiuréia (HU) constitui o avanço mais importante no tratamento da anemia falciforme (AF) por prevenir complicações e aumentar a qualidade de vida dos pacientes. Entretanto, alguns aspectos do tratamento com HU permanecem obscuros, incluindo a sua ação e potencial toxicidade em outras células sanguíneas, tais como neutrófilos. Este estudo utilizou a mensuração da lactato desidrogenase (LDH) e do metil tiazoltetrazólio (MTT) e o ensaio do cometa para investigar a citotoxicidade e índice de dano (ID) ao DNA em neutrófilos de pacientes com AF em uso do medicamento. Nos ensaios de LDH e MTT, observou-se além de ausência de toxicidade, uma ação citoprotetora no grupo de pacientes tratados, Grupo SSHU (n=21, 11 mulheres e 10 homens, com idades entre 19-63 anos), quando comparados aos pacientes sem tratamento, Grupo SS (n=20, 13 mulheres e 07 homens, 18-69 anos), e grupo de indivíduos saudáveis (AA) usado como controle (n=52, 28 mulheres e 24 homens, 19-60 anos), com redução significativa (p<0,001) na atividade de LDH e aumento no percentual de células viáveis pelo MTT (p<0,001). Entretanto, o grupo SSHU apresentou valores de ID significativamente elevados (49,57±6,0 U/A), quando comparados ao grupo AA (7,43 ± 0,94U/A) e grupo SS (22,73 ±5,58 U/A) (p<0,0001), especialmente quando tratados por períodos mais longos (>20 meses), demonstrando que apesar dos efeitos citoprotetores quanto à viabilidade celular, o uso da HU pode induzir lesão ao DNA de neutrófilos.

Derivados quiolínicos: compustos sintéticos con propiedades antitumorales sobre líneas de cáncer humano / Quinolinic derivates: synthetic compounds with antitumor proprieties against human line cell cancer

El cáncer es una enfermedad multifactorial caracterizada por anormalidad en el crecimiento celular provocado por factores ambientales y múltiples cambios en la expresión de los genes. El cáncer de mama representa una de las enfermedades que más afecta a la población femenina mundial. Actualmente, se ha mostrado un auge en las investigaciones sobre compuestos extraídos de diversas plantas y origen sintético con posibles agentes antitumorales, entre los que se pueden nombrar los derivados sintéticos de quinolinas. Realizado estudio del grupo sintético indeno (2,1-c) quinolinas y sus efectos citotóxicos sobre líneas de cáncer de mama SKBr3 yMCF-7. La evaluación de la actividad citotóxica de estos compuestos fue determinada a través del método del MTT, se calcularon los valores de concentración inhibitoria, los índices de selectividad y combinaciones con taxanos. Los resultados obtenidos muestran una alta selectividad de los compuestos sintéticos y natural evaluados hacia las líneas celulares MCF-7 y SKBr3 con respecto a las células control (fibroblastos dérmicos humanos); además de una interacción de tipo potenciación en la combinación de un derivado quinolínico y la drogataxano. Con todo esto se puede inferir que solo ciertos compuestos que tuvieron modificaciones químicas realizadas sobre el compuesto patrón del grupo sintético indeno (2,1-c) quinolinas mostraron una inhibición sobre la viabilidad celular sobre las líneas tumorales SKBr3 y MCF-7, conforme aumenta la dosis de los compuestos, además de evidenciar la efectividad de compuestos derivados de quinolinas como moléculas con características antineoplásicas

The cancer is a multifactorial disease characterized by abnormal cell growth caused by environmental factors and for multiple changes in the gene expression. The breast cancer is a disease that affects the female population in worldwide. Currently, there is shown an increment in the research on compounds that we extracted from various plants and synthetic origin with potential antitumor agents, among which can be namedthe synthetic derivatives of the quinolines. A study was performed on an group of synthetic indeno (2,1-c) quinolines and their cytotoxic effects on breast cancer cell lines SKBR3 and MCF-7 was performed. The evaluation of the cytotoxic activity of these compounds was determined through the MTT method; we were calculated inhibitory concentration values, the rates of selectivity and the drug combinations with taxanes. The results show a high selectivity of the natural and the synthetic compounds evaluated towards the cell lines MCF-7 and SKBR3 cells with respect to control (dermal fibroblasts human), besides an enhancement type interaction in the combination of a drug derivative and the quinolinic taxanes. With all this we can infer that only certain compounds had chemical modifications performed on the composite pattern of synthetic group indeno (2, 1-c) quinolines showed an inhibition on cell viability on tumor cell lines SKBR3 and MCF-7, with increasing dose of the compounds, also showing the effectiveness of compounds derived from the quinolines as molecules with the antineoplastic properties and his characteristics

Avaliação in vitro da citotoxicidade de ligas metálicas utilizadas para a confecção de próteses dentárias / In vitro evaluation of the cytotoxicity of metal alloys used to manufacture dental prosthesis

Metais são amplamente utilizados em procedimentos de reabilitação oral. Esses materiais entram em contato com estruturas biológicas quando utilizados na cavidade bucal e podem desenvolver citotoxicidade. O objetivo deste estudo foi avaliar a citotoxicidade de diferentes ligas metálicas utilizadas para a confecção de próteses odontológicas. Quatro ligas metálicas foram avaliadas: níquel-cromo (grupo 1), cobre-alumínio (grupo 2), cobalto-cromo (grupo 3) e prata (grupo 4). Inicialmente, os corpos de prova foram preparados utilizando uma matriz padronizada e foram imersos em meio de cultura de células durante 24h para a possível liberação de toxinas, sendo então, colocados em contato com os fibroblastos L929 nos períodos de 24 e 48h. A viabilidade celular foi avaliada em espectrofotômetro. Três grupos adicionais foram avaliados: controle de células grupo C+ (controle positivo), corpos de prova em amálgama e C- (controle negativo), fios de aço em contato com as células. Os dados foram submetidos aos testes estatísticos ANOVA e Tukey. Os resultados mostraram citotoxicidade nos grupos 1 (níquel-cromo), 2 (cobre-alumínio) e 4 (prata) após 24h; estes grupos apresentaram diferença estatística significante entre CC (controle de células) e C- (controle negativo) (p<0.05). Após 48h, a citotoxicidade estava ausente no grupo 1. Todavia, os grupos 2 e 4 ainda apresentaram alta citotoxicidade (p<0.05). A liga de níquel- -cromo apresentou citotoxicidade às células fibroblásticas nas primeiras 24h, enquanto a liga de prata e de cobre alumínio apresentou nas 24 e 48h, sendo maior para cobre-alumínio. A liga de cobalto-cromo não apresentou citotoxicidade

Metals are still widely used in oral rehabilitation procedures. These materials are in contact with biological structures when used within the oral cavity and can develop cytotoxic effects. This study aimed to evaluate the cytotoxicity of different alloys used when fabricating dental prostheses. Four alloys were evaluated: nickel-chromium (group 1), copper-aluminum (group 2), cobalt-chromium (group 3), and silver (group 4). Initially, test bodies were prepared using a standardization matrix. Next, the test bodies were immersed in culture media for 24h to release possible toxins. The medium was then placed in contact with L929 cells for 24 and 48h. The cells were then stained and analyzed for viability in a spectrophotometer. Three additional groups were evaluated: control cell group C+ (positive control), consisting of an amalgam test body; and C- (negative control), stainless steel wire in contact with the cells. The data were analyzed by ANOVA and the Tukey test. Analysis of the results verified cytotoxicity in groups 1 (nickel-chromium), 2 (copper-aluminum) and 4 (silver alloy) at 24h. These groups presented statistically significant differences with the CC (control cell) and C- groups (negative control) (p<0.05). After 48h, cytotoxicity was absent in group 1. However, groups 2 and 4 still presented high cytotoxicity (p<0.05). Nickel-chromium alloy presented cytotoxicity for fibroblast cells in the first 24h, while silver and copper-aluminum alloys showed at 24 and 48h, which was greater for copper-aluminum alloy. Cobalt-chromium alloy presented no cytotoxicity

A chimeric antibody to L1 cell adhesion molecule shows therapeutic effect in an intrahepatic cholangiocarcinoma model

Intrahepatic cholangiocarcinoma (ICC), a malignant tumor derived from the intrahepatic bile duct epithelium, has a poor prognosis and is refractory to conventional chemotherapy and radiation therapy. Thus, there is an urgent need to develop new effective therapeutic strategies for this disease. We previously found that L1 cell adhesion molecule (L1CAM) plays an important role in tumor progression of ICC, and we generated a murine mAb, A10-A3 (IgG1), that binds to the Ig1 domain of L1CAM. In the present study, we further characterized A10-A3, constructed a chimeric A10-A3 antibody (cA10-A3) containing the constant regions of human IgG1, and evaluated the therapeutic potential in a human ICC xenograft nude mice model. The affinities (K D) of A10-A3 and cA10-A3 for soluble L1CAM were 1.8 nM and 1.9 nM, respectively, as determined by competition ELISA. A10-A3 inhibited L1CAM homophilic binding and was slowly internalized into the tumor cells, but it did not significantly inhibit proliferation of ICC cells in vitro. cA10-A3 mediated antibody-dependent cell-mediated cytotoxicity in vitro and displayed anti-tumor activity in the ICC animal model. These results suggest that the humanized A10-A3 antibody may have potential as an anticancer agent for the treatment of ICC.

Associação de polimorfismos e expressão gênica de marcadores de apoptose ligados carcinogênese cervical induzida pelo hpv em mulheres infectadas ou não pelo HIV / Association of polymorphisms and gene expression of apoptosis markers linked cervical carcinogenesis induced hpv in women infected with HIV or not

A frequência observada desses SNPs estavam em equilíbrio de Hardy–Weinberg nos casos e controles. Nenhuma diferença estatística na frequência genotípica e alélica do polimorfismo de BCL-2 (-938C>A) foi observada entre casos e controles. Contudo, uma forte associação foi identificada na freqüência do genótipo GG de BAX (-248G>A) entre os casos e controles. Quando os grupos de LSIL e HSIL foram comparados, nenhuma diferença estatística foi observada, indicando que o genótipo GG pode influenciar no aparecimento de lesões cervicais, mas não na gravidade da doença. Analisamos a expressão de RNAm de genes associados a apoptose (BAX, BCL2, p53 e pRb) e citotoxicidade (perforina e Fas). Quando a expressão de RNAm de Perforina e Fas foram avaliadas, diferença estatística foi observadas em pacientes portadoras de NIC III e câncerObservamos diferença estatística entre os controles e todos os casos, quando analisamos a expressão de RNAm de p53, mas não de pRb, sendo observada diferença apenas nos controles e NIC II e III. Na analise de RNAm de BCL2, notamos uma baixa expressão no grupo de câncer quando comparado com NIC II, III e controle. Em conclusão, a infecção pelo HIV pode induzir redução na degranulação de células inflamatórias, corroborando para a progressão de infecção pelo HPV, e que carreadoras do alelo G na região promotora de BAX (-248G>A) pode estar associado ao desenvolvimento de NIC quando comparado com as carreadoras do alelo A, possuindo papel protetor; contudo o alelo G não está correlacionado com a gravidade da doença...

Cell-mediated cytotoxicity plays an important role in the regulation of HPVinfection, and in tumor formation. Apoptosis is regulated by different pathways involving a number of genes that either promote (BAX gene)or inhibit (BCL2 gene) the cell death. We evaluated the presence of cervical inflammatory cells, apoptotic (Bax, Bcl-2, FasL, NOS2, perforin) markers and the degranulating expressing cell marker (CD107a) from HPV and HPV/HIV women. Higher per centage of cervical CD4 + and CD8 + T cells were observed in both groups, with lower CD4+ T cells count observed in HPV/HIV women. There were few FasL, Bax and Bcl - 2 inflammatory cervical expressing cells in both groups. NOS2 expression was present especially in the epithelium basal layer on keratinocytes in both groups. Perforin was identified in few cervical cells. However, CD107a was detected in the epithelium basal layer and stroma, meanly on HPV women. We determined whether the SNPs of BCL2(-938C>A) and BAX ( - 248G>A) promoters are associated with risk of cervical intraepithelial neoplasia (CIN) outcome. The observed genotype frequencies of these SNPs were in agreement with Hardy – Weinberg equilibrium in cases and controlgroups. No statistical difference in genotype and allelic frequency ofBCL - 2 ( - 938C>A) polymorphism was observed between all cases and control groups. However, a strong association was identified in GG genotype frequency of BAX ( - 248G>A) polymorphism between cases and controls. When LSIL an d HSILgroups were compared, no statistical difference was observed, indicating thatGG genotype may influence the risk of CIN but not the lesion severity. Weanalyzed the mRNA expression of apoptosis - associated genes (BAX, BCL2, p53 and pRb ) and cytotoxicity -related genes (perforin and Fas). When Fas and Perforin mRNA expressions were evaluated, the statistical difference betweenCIN III and cancer was seen...