Comparison of senescence progression in mesenchymal cells from human umbilical cord walls measured by immunofluorescence and flow cytometry of p16 and p21.

OBJECTIVE: To follow the expansion of mesenchymal stem cells from umbilical cords by two classic senescence markers, p16 (INK4A) and p21 (CDKN1A), using practical, fast, and less expensive methods than the gold standard Western blotting technique, to evaluate its applicability in the laboratory. METHODS: Mesenchymal stem cells from umbilical cords were isolated from Wharton's jelly and, after quality control, morphological and immunophenotypic characterization by flow cytometry, were expanded in culture until coming close to cell cycle arrest (replicative senescence). RESULTS: A comparison was made between young cells, at passage 5, and pre-senescent cells, at passage 10, evaluating the protein expression of the classic cell senescence markers p16 and p21, comparing the results obtained by Western blotting with those obtained by flow cytometry and indirect immunofluorescence. CONCLUSION: Follow-up of cell cultures, through indirect p16 immunofluorescence, allows the identification of mesenchymal stem cells from umbilical cord cultures at risk of reaching replicative senescence.

Cytokine and Chemokine Concentration in the Tear of Patients with Age-Related Cataract.

PURPOSE: The present study measured 41 soluble mediators in the tear of 19 patients with age-related cataract and 32 healthy adults as controls. MATERIALS AND METHODS: This was a prospective, case-control study in which, using multiple immunoassays, we measured in tear samples the following molecules: EGF, FGF-2, Eotaxin, TGF-α, G-CSF, Flt-3L, GM-CSF, Fractalkine, IFN-α2, IFN-γ, GRO, IL-10, MCP-3, IL-12p40, MDC, IL-12p70, PDGF-AA, IL-13, PDGF-AB/BB, IL-15, sCD40L, IL-17a, IL-1ra, IL-1α, IL-9, IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP-1α, MIP-1ß, RANTES, TNFα, TNFß, VEGF. Statistical analyses were done by multiple adjusted models and p values were corrected by the Benjamini and Hochberg method. RESULTS: We did not find significant differences in the amount of the tested molecules in the tear fluid between cataract patients and controls. Correlation analyses relative to age were carried out for both groups. Analysis of MCP-1 tear levels revealed a direct correlation with age for normal healthy controls as well as for cataract patients. But IL-6 tear levels correlated with age only in the group of cataract patients. In addition, IL1-ra tear levels correlated with cataract nuclear grade; higher grades were associated with higher IL-1ra concentrations. CONCLUSIONS: Our results suggest that ocular aging is accompanied by increased production of IL-6 and MCP-1, which can be measured in tear fluid. ABBREVIATIONS: AMD: Age-Related Macular Degeneration; EGF: Epidermal growth factor; Eotaxin: Eosinophil chemotactic proteins; FasL: Fas ligand; FGF-2: Basic fibroblast growth factor 2; Flt-3L: Fms related tyrosine kinase 3 ligand; G-CSF: Granulocyte colony stimulating factor; GM-CSF: Granulocyte macrophage colony stimulating factor; GRO: Growth regulated protein; HGF: Human growth factor; ICAM-1: Intercellular adhesion molecule-1; IFNα2: Interferon alpha 2; IFNγ: Interferon gamma; IL: Interleukin; IL-1ra: Interleukin-1 receptor antagonist; IL-12p40: Interleukin-12 subunit p40; IL-12p70: Interleukin-12 subunit p70; IP-10: Interferon gamma-induced protein 10; MCP-1: Monocyte chemotactic protein 1; MCP-3: Monocyte chemotactic protein 3; MDC: Macrophage derived chemokine; MIG: Monokine induced by gamma interferon; MIP-1α: Macrophage inflammatory proteins 1 alpha; MIP-1ß: Macrophage inflammatory proteins 1 beta; MMPs: Matrix metalloproteinases; MMP-9: Matrix metalloproteinase 9; PAI1: Plasminogen activator inhibitor 1; PDGF-AA: Platelet-derived growth factor subunit AA; PDGF-AB/BB: Platelet-derived growth factor subunit AB and BB; PIGF: Placenta growth factor; RANTES: Regulated on activation, normal T cell expressed and secreted; SAA: Serum amyloid A; sCD40L: Soluble CD40 ligand; sTNF-RII: Soluble tumor necrosis factor receptor; TBUT Tear breakup time; TGF-α: Transforming growth factor alpha; TGF-ß: Transforming growth factor beta; TNFα: Tumor necrosis factor alpha; TNFß: Tumor necrosis factor beta; VCAM: Vascular cell adhesion molecule; VEGF: Vascular endothelium growth factor.

Comparison of senescence progression in mesenchymal cells from human umbilical cord walls measured by immunofluorescence and flow cytometry of p16 and p21 / Comparação da progressão da senescência em células mesenquimais de parede de cordão umbilical humano medida por imunofluorescência e citometria de fluxo de p16 e p21

ABSTRACT Objective To follow the expansion of mesenchymal stem cells from umbilical cords by two classic senescence markers, p16 (INK4A) and p21 (CDKN1A), using practical, fast, and less expensive methods than the gold standard Western blotting technique, to evaluate its applicability in the laboratory. Methods Mesenchymal stem cells from umbilical cords were isolated from Wharton's jelly and, after quality control, morphological and immunophenotypic characterization by flow cytometry, were expanded in culture until coming close to cell cycle arrest (replicative senescence). Results A comparison was made between young cells, at passage 5, and pre-senescent cells, at passage 10, evaluating the protein expression of the classic cell senescence markers p16 and p21, comparing the results obtained by Western blotting with those obtained by flow cytometry and indirect immunofluorescence. Conclusion Follow-up of cell cultures, through indirect p16 immunofluorescence, allows the identification of mesenchymal stem cells from umbilical cord cultures at risk of reaching replicative senescence.

RESUMO Objetivo Acompanhar a expansão de células-tronco mesenquimais de cordão umbilical por dois marcadores clássicos de senescência, p16 (INK4A) e p21 (CDKN1A), usando métodos práticos, rápidos e com custo menor do que a técnica padrão-ouro de Western blotting, para avaliar sua aplicabilidade em laboratório. Métodos Células-tronco mesenquimais de cordão umbilical foram isoladas da geleia de Wharton e, após controle de qualidade e caracterização morfológica e imunofenotípica por citometria de fluxo, foram expandidas em cultura, até chegarem próximas à parada do ciclo celular (senescência replicativa). Resultados Foi feita a comparação entre células jovens, na passagem 5, e células pré-senescentes, na passagem 10, avaliando a expressão proteica dos marcadores clássicos de senescência celular p16 e p21, comparando os resultados obtidos por Western blotting com os obtidos por citometria de fluxo e imunofluorescência indireta. Conclusão O seguimento de culturas celulares, por meio da imunofluorescência indireta de p16, permite identificar as culturas de células-tronco mesenquimais de cordão umbilical em risco de atingirem a senescência replicativa.

Profile of potential organ donors and factors related to donation and non- donation of organs in an Organ Procurement Service. / Perfil dos potenciais doadores de órgãos e fatores relacionados à doação e a não doação de órgãos de uma Organização de Procura de Órgãos.

OBJECTIVE: to characterize the profile of potential and effective organ donors, and to identify the factors related to non-donation. METHODS: the data was collected from the Organization of Organ Donation forms during the period of January 2013 to April 2018. The statistical analysis was done by the Biostatistic Service of the School of Medical Sciences of UNICAMP. RESULTS: the study analyzed 1,772 potential donors; the male gender was predominant (57.39%). Vascular injuries (n=996) were the main cause of brain death. The family refusal (42.8%) was the leading cause of non-donation of organs. There was statistical difference between donors and non-donors in regard to the mean age and mean systolic blood pressure. There was also a relationship between the donation of organs and the absence of diabetes and smoking. CONCLUSION: the majority of effective organ donors were young males. The main causes of brain death (BD) and family refusal of organ donation were cerebrovascular disorder and no desire to donate organs after death, respectively. Alcoholics and males were more frequently found in traumatic causes of BD. Therefore, initiatives for population awareness and discussion among the families regarding organ donation can lead to increase the number of effective organ donors.

OBJETIVO: caracterizar o perfil dos potenciais e dos efetivos doadores de órgãos, e identificar os fatores relacionados a não efetivação da doação. MÉTODOS: estudo retrospectivo transversal com coleta de dados das fichas da Organização de Procura de Órgãos do Hospital das Clínicas da Unicamp, referente ao período de janeiro de 2013 a abril de 2018. RESULTADOS: o estudo contou com 1.772 potenciais doadores; predominou-se o sexo masculino (57,39%) e o evento vascular (n=996) foi a principal causa de morte encefálica. A recusa familiar (42,8%) foi o motivo mais comum para não doação de órgãos. Houve diferença estatística entre doadores e não doadores quanto à média de idade e pressão arterial sistólica, assim como houve relação entre a doação de órgãos e a ausência de diabetes e tabagismo. CONCLUSÃO: a maioria dos doadores efetivos foi do sexo masculino e jovem. As principais causas de morte encefálica e de recusa familiar foram, respectivamente, vasculares e não manifestação da vontade de ser doador após a morte. O etilismo foi mais presente nas causas traumáticas, assim como, o sexo masculino. Assim, iniciativas de conscientização populacional e abertura de discussão dentro da família sobre o ato da doação contribuem para o aumento do índice de doadores efetivos.

Correction: CD28 deficiency leads to accumulation of germinal-center independent IgM+ experienced B cells and to production of protective IgM during experimental malaria.

[This corrects the article DOI: 10.1371/journal.pone.0202522.].

Perfil dos potenciais doadores de órgãos e fatores relacionados à doação e a não doação de órgãos de uma Organização de Procura de Órgãos / Profile of potential organ donors and factors related to donation and non- donation of organs in an Organ Procurement Service

RESUMO Objetivo: caracterizar o perfil dos potenciais e dos efetivos doadores de órgãos, e identificar os fatores relacionados a não efetivação da doação. Métodos: estudo retrospectivo transversal com coleta de dados das fichas da Organização de Procura de Órgãos do Hospital das Clínicas da Unicamp, referente ao período de janeiro de 2013 a abril de 2018. Resultados: o estudo contou com 1.772 potenciais doadores; predominou-se o sexo masculino (57,39%) e o evento vascular (n=996) foi a principal causa de morte encefálica. A recusa familiar (42,8%) foi o motivo mais comum para não doação de órgãos. Houve diferença estatística entre doadores e não doadores quanto à média de idade e pressão arterial sistólica, assim como houve relação entre a doação de órgãos e a ausência de diabetes e tabagismo. Conclusão: a maioria dos doadores efetivos foi do sexo masculino e jovem. As principais causas de morte encefálica e de recusa familiar foram, respectivamente, vasculares e não manifestação da vontade de ser doador após a morte. O etilismo foi mais presente nas causas traumáticas, assim como, o sexo masculino. Assim, iniciativas de conscientização populacional e abertura de discussão dentro da família sobre o ato da doação contribuem para o aumento do índice de doadores efetivos.

ABSTRACT Objective: to characterize the profile of potential and effective organ donors, and to identify the factors related to non-donation. Methods: the data was collected from the Organization of Organ Donation forms during the period of January 2013 to April 2018. The statistical analysis was done by the Biostatistic Service of the School of Medical Sciences of UNICAMP. Results: the study analyzed 1,772 potential donors; the male gender was predominant (57.39%). Vascular injuries (n=996) were the main cause of brain death. The family refusal (42.8%) was the leading cause of non-donation of organs. There was statistical difference between donors and non-donors in regard to the mean age and mean systolic blood pressure. There was also a relationship between the donation of organs and the absence of diabetes and smoking. Conclusion: the majority of effective organ donors were young males. The main causes of brain death (BD) and family refusal of organ donation were cerebrovascular disorder and no desire to donate organs after death, respectively. Alcoholics and males were more frequently found in traumatic causes of BD. Therefore, initiatives for population awareness and discussion among the families regarding organ donation can lead to increase the number of effective organ donors.

CD28 deficiency leads to accumulation of germinal-center independent IgM+ experienced B cells and to production of protective IgM during experimental malaria.

Protective immunity to blood-stage malaria is attributed to Plasmodium-specific IgG and effector-memory T helper 1 (Th1) cells. However, mice lacking the costimulatory receptor CD28 (CD28KO) maintain chronic parasitemia at low levels and do not succumb to infection, suggesting that other immune responses contribute to parasite control. We report here that CD28KO mice develop long-lasting non-sterile immunity and survive lethal parasite challenge. This protection correlated with a progressive increase of anti-parasite IgM serum levels during chronic infection. Serum IgM from chronically infected CD28KO mice recognize erythrocytes infected with mature parasites, and effectively control Plasmodium infection by promoting parasite lysis and uptake. These antibodies also recognize autoantigens and antigens from other pathogens. Chronically infected CD28KO mice have high numbers of IgM+ plasmocytes and experienced B cells, exhibiting a germinal-center independent Fas+GL7-CD38+CD73- phenotype. These cells are also present in chronically infected C57BL/6 mice although in lower numbers. Finally, IgM+ experienced B cells from cured C57BL/6 and CD28KO mice proliferate and produce anti-parasite IgM in response to infected erythrocytes. This study demonstrates that CD28 deficiency results in the generation of germinal-center independent IgM+ experienced B cells and the production of protective IgM during experimental malaria, providing evidence for an additional mechanism by which the immune system controls Plasmodium infection.

CD28 deficiency leads to accumulation of germinal-center independent IgM+ experienced B cells and to production of protective IgM during experimental malaria

Protective immunity to blood-stage malaria is attributed to Plasmodium-specific IgG and effector-memory T helper 1 (Th1) cells. However, mice lacking the costimulatory receptor CD28 (CD28KO) maintain chronic parasitemia at low levels and do not succumb to infection, suggesting that other immune responses contribute to parasite control. We report here that CD28KO mice develop long-lasting non-sterile immunity and survive lethal parasite challenge. This protection correlated with a progressive increase of anti-parasite IgM serum levels during chronic infection. Serum IgM from chronically infected CD28KO mice recognize erythrocytes infected with mature parasites, and effectively control Plasmodium infection by promoting parasite lysis and uptake. These antibodies also recognize autoantigens and antigens from other pathogens. Chronically infected CD28KO mice have high numbers of IgM+ plasmocytes and experienced B cells, exhibiting a germinal-center independent Fas+GL7-CD38+CD73- phenotype. These cells are also present in chronically infected C57BL/6 mice although in lower numbers. Finally, IgM+ experienced B cells from cured C57BL/6 and CD28KO mice proliferate and produce anti-parasite IgM in response to infected erythrocytes. This study demonstrates that CD28 deficiency results in the generation of germinal-center independent IgM+ experienced B cells and the production of protective IgM during experimental malaria, providing evidence for an additional mechanism by which the immune system controls Plasmodium infection

CD28 deficiency leads to accumulation of germinal-center independent IgM+ experienced B cells and to production of protective IgM during experimental malaria

Protective immunity to blood-stage malaria is attributed to Plasmodium-specific IgG and effector-memory T helper 1 (Th1) cells. However, mice lacking the costimulatory receptor CD28 (CD28KO) maintain chronic parasitemia at low levels and do not succumb to infection, suggesting that other immune responses contribute to parasite control. We report here that CD28KO mice develop long-lasting non-sterile immunity and survive lethal parasite challenge. This protection correlated with a progressive increase of anti-parasite IgM serum levels during chronic infection. Serum IgM from chronically infected CD28KO mice recognize erythrocytes infected with mature parasites, and effectively control Plasmodium infection by promoting parasite lysis and uptake. These antibodies also recognize autoantigens and antigens from other pathogens. Chronically infected CD28KO mice have high numbers of IgM+ plasmocytes and experienced B cells, exhibiting a germinal-center independent Fas+GL7-CD38+CD73- phenotype. These cells are also present in chronically infected C57BL/6 mice although in lower numbers. Finally, IgM+ experienced B cells from cured C57BL/6 and CD28KO mice proliferate and produce anti-parasite IgM in response to infected erythrocytes. This study demonstrates that CD28 deficiency results in the generation of germinal-center independent IgM+ experienced B cells and the production of protective IgM during experimental malaria, providing evidence for an additional mechanism by which the immune system controls Plasmodium infection

BCR-ABL1-induced downregulation of WASP in chronic myeloid leukemia involves epigenetic modification and contributes to malignancy.

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the BCR-ABL1 tyrosine kinase (TK). The development of TK inhibitors (TKIs) revolutionized the treatment of CML patients. However, TKIs are not effective to those at advanced phases when amplified BCR-ABL1 levels and increased genomic instability lead to secondary oncogenic modifications. Wiskott-Aldrich syndrome protein (WASP) is an important regulator of signaling transduction in hematopoietic cells and was shown to be an endogenous inhibitor of the c-ABL TK. Here, we show that the expression of WASP decreases with the progression of CML, inversely correlates with the expression of BCR-ABL1 and is particularly low in blast crisis. Enforced expression of BCR-ABL1 negatively regulates the expression of WASP. Decreased expression of WASP is partially due to DNA methylation of the proximal WASP promoter. Importantly, lower levels of WASP in CML advanced phase patients correlate with poorer overall survival (OS) and is associated with TKI response. Interestingly, enforced expression of WASP in BCR-ABL1-positive K562 cells increases the susceptibility to apoptosis induced by TRAIL or chemotherapeutic drugs and negatively modulates BCR-ABL1-induced tumorigenesis in vitro and in vivo. Taken together, our data reveal a novel molecular mechanism that operates in BCR-ABL1-induced tumorigenesis that can be used to develop new strategies to help TKI-resistant, CML patients in blast crisis (BC).

Intrinsic Variability Present in Wharton's Jelly Mesenchymal Stem Cells and T Cell Responses May Impact Cell Therapy.

Wharton's jelly mesenchymal stem cells (WJ-MSC) exhibit immunomodulatory effects on T cell response. WJ-MSC are easy to collect, process, and proliferate rapidly in culture, but information on the variability of individual cell samples impacting upon in vitro expansion, immunomodulatory potential, and aging processes is still lacking. We propose to evaluate the immunomodulatory cytokine profile and capacity to inhibit T cell proliferation of WJ-MSC progressing to replicative senescence in order to analyze if expected responses are affected. Our results show that the gene expression of immunomodulatory molecules varied among samples with no specific pattern present. In coculture, all WJ-MSC were capable of inhibiting mitogen-activated CD3+ T cell proliferation, although to different degrees, and each PBMC responded with a different level of inhibition. Thus, we suggest that each WJ-MSC displays unique behavior, differing in patterns of cytokine mRNA expression and immunomodulatory capacity. We believe that variability between samples may play a role in the effectiveness of WJ-MSC employed therapeutically.

Novel CD28 antagonist mPEG PV1-Fab' mitigates experimental autoimmune uveitis by suppressing CD4+ T lymphocyte activation and IFN-γ production.

Autoimmune Uveitis is an important chronic inflammatory disease and a leading cause of impaired vision and blindness. This ocular autoimmune disorder is mainly mediated by T CD4+ lymphocytes poising a TH1 phenotype. Costimulatory molecules are known to play an important role on T cell activation and therefore represent interesting therapeutical targets for autoimmune disorders. CD28 is the prototypical costimulatory molecule for T lymphocytes, and plays a crucial role in the initiation, and maintenance of immune responses. However, previous attempts to use this molecule in clinical practice achieved no success. Thus, we evaluated the efficacy of mPEG PV1-Fab' (PV1), a novel selective CD28 antagonist monovalent Fab fragment in the treatment of Experimental Autoimmune Uveitis (EAU). Here, we showed that PV1 treatment decreases both average disease score and incidence of EAU. A decrease in the activation profile of both T CD4+ and T CD8+ eye-infiltrating lymphocytes was evidenced. In the periphery, T CD4+ cells from PV1-treated mice also showed a decrease in their activation status, with reduced expression of CD69, CD25, and PD-1 molecules. This suppression was not dependent on Treg cells, as both their frequency and absolute number were lower in PV1-treated mice. In addition, frequency of CD4+IFN-γ+ T cells was significantly lower in PV1-treated group, but not of IL-17-producing T cells. Moreover, after specific restimulation, PV1 blockade selectively blocked IFN-γ production by CD4+ lymphocytes Taken together, our data suggest that mPEG PV1-Fab' acts mainly on IFN-γ-producing CD4+ T cells and emphasize that this specific CD28 blockade strategy is a potential specific and alternative tool for the treatment of autoimmune disorders in the eye.

Guidelines for the assessment and acceptance of potential brain-dead organ donors. / Diretrizes para avaliação e validação do potencial doador de órgãos em morte encefálica.

Organ transplantation is the only alternative for many patients with terminal diseases. The increasing disproportion between the high demand for organ transplants and the low rate of transplants actually performed is worrisome. Some of the causes of this disproportion are errors in the identification of potential organ donors and in the determination of contraindications by the attending staff. Therefore, the aim of the present document is to provide guidelines for intensive care multi-professional staffs for the recognition, assessment and acceptance of potential organ donors.

Analysis of cytokine profile and growth factors in platelet-rich plasma obtained by open systems and commercial columns.

OBJECTIVE:: To evaluate growth factors and cytokines in samples of platelet-rich plasma obtained by three different centrifugation methods. METHODS:: Peripheral blood of six individuals with no hematological diseases, aged 18 to 68 years, was drawn to obtain platelet-rich plasma, using the open method and commercial columns by Medtronic and Biomet. The products obtained with the different types of centrifugation were submitted to laboratory analysis, including pro-inflammatory cytokines and chemokines by flow cytometry assays, the concentration of fibroblast growth factors-2 (FGF-2) and transforming growth factor-beta1 (TGF-ß1). RESULTS:: The diverse separation methods generated systematically different profiles regarding number of platelets and leukocytes. The Medtronic system yielded a product with the highest concentration of platelets, and the open method, with the lowest concentration of platelets. The results of cytokine analysis showed that the different types of centrifugation yielded products with high concentrations of interleukin 8, interleukin 1ß. The open system resulted in a product with high levels of interleukin 6. Other cytokines and chemokines measured were similar between systems. The product obtained with the open method showed higher levels of TGF-ß1 in relation to other systems and low FGF-2 levels. CONCLUSION:: The formed elements, growth factors and cytokines in samples of platelet-rich plasma varied according to the centrifugation technique used. OBJETIVO:: Avaliar fatores de crescimento e citocinas em amostras de plasma rico em plaquetas obtidas por três diferentes métodos de centrifugação. MÉTODOS:: Foi coletado sangue periférico de seis indivíduos, sem doença hematológica, com idades entre 18 e 68 anos, para obtenção de plasma rico em plaquetas, utilizando o método aberto e sistemas comerciais das empresas Medtronic e Biomet. Os produtos obtidos com os diferentes tipos de centrifugação foram submetidos às análises laboratoriais, incluindo citocinas próinflamatórias e quimiocinas, por meio de ensaios de citometria de fluxo, concentração do fator de crescimento fibroblástico-2 (FGF-2) e fator de crescimento transformador-beta1 (TGF-ß1). RESULTADOS:: As diferentes centrifugações geraram perfis sistematicamente diferentes referentes ao número de plaquetas e de leucócitos. O sistema da Medtronic originou produto com a maior concentração de plaquetas, e o método aberto com a menor concentração de plaquetas. Os resultados da análise de citocinas demonstraram que os diferentes tipos de centrifugação originaram produtos com elevadas concentrações de interleucina 8 e interleucina 1ß. O sistema aberto resultou em produto com elevados níveis de interleucina 6. As demais citocinas e quimiocinas mensuradas foram similares entre os sistemas. O produto obtido com o método aberto apresentou níveis superiores de TGF-ß1 em relação aos demais sistemas e reduzidos níveis de FGF-2. CONCLUSÃO:: Os elementos figurados, fatores de crescimento e citocinas, em amostras de plasma rico em plaquetas, variaram conforme a técnica de centrifugação utilizada.

Diretrizes para avaliação e validação do potencial doador de órgãos em morte encefálica / Guidelines for the assessment and acceptance of potential brain-dead organ donors

RESUMO O transplante de órgãos é a única alternativa para muitos pacientes portadores de algumas doenças terminais. Ao mesmo tempo, é preocupante a crescente desproporção entre a alta demanda por transplantes de órgãos e o baixo índice de transplantes efetivados. Dentre as diferentes causas que alimentam essa desproporção, estão os equívocos na identificação do potencial doador de órgãos e as contraindicações mal atribuídas pela equipe assistente. Assim, o presente documento pretende fornecer subsídios à equipe multiprofissional da terapia intensiva para o reconhecimento, a avaliação e a validação do potencial doador de órgãos.

ABSTRACT Organ transplantation is the only alternative for many patients with terminal diseases. The increasing disproportion between the high demand for organ transplants and the low rate of transplants actually performed is worrisome. Some of the causes of this disproportion are errors in the identification of potential organ donors and in the determination of contraindications by the attending staff. Therefore, the aim of the present document is to provide guidelines for intensive care multi-professional staffs for the recognition, assessment and acceptance of potential organ donors.

Analysis of cytokine profile and growth factors in platelet-rich plasma obtained by open systems and commercial columns / Análise do perfil de citocinas e fatores de crescimento em plasma rico em plaquetas obtido por meio das metodologias do sistema aberto e colunas

ABSTRACT Objective: To evaluate growth factors and cytokines in samples of platelet-rich plasma obtained by three different centrifugation methods. Methods: Peripheral blood of six individuals with no hematological diseases, aged 18 to 68 years, was drawn to obtain platelet-rich plasma, using the open method and commercial columns by Medtronic and Biomet. The products obtained with the different types of centrifugation were submitted to laboratory analysis, including pro-inflammatory cytokines and chemokines by flow cytometry assays, the concentration of fibroblast growth factors-2 (FGF-2) and transforming growth factor-beta1 (TGF-β1). Results: The diverse separation methods generated systematically different profiles regarding number of platelets and leukocytes. The Medtronic system yielded a product with the highest concentration of platelets, and the open method, with the lowest concentration of platelets. The results of cytokine analysis showed that the different types of centrifugation yielded products with high concentrations of interleukin 8, interleukin 1β. The open system resulted in a product with high levels of interleukin 6. Other cytokines and chemokines measured were similar between systems. The product obtained with the open method showed higher levels of TGF-β1 in relation to other systems and low FGF-2 levels. Conclusion: The formed elements, growth factors and cytokines in samples of platelet-rich plasma varied according to the centrifugation technique used.

RESUMO Objetivo: Avaliar fatores de crescimento e citocinas em amostras de plasma rico em plaquetas obtidas por três diferentes métodos de centrifugação. Métodos: Foi coletado sangue periférico de seis indivíduos, sem doença hematológica, com idades entre 18 e 68 anos, para obtenção de plasma rico em plaquetas, utilizando o método aberto e sistemas comerciais das empresas Medtronic e Biomet. Os produtos obtidos com os diferentes tipos de centrifugação foram submetidos às análises laboratoriais, incluindo citocinas próinflamatórias e quimiocinas, por meio de ensaios de citometria de fluxo, concentração do fator de crescimento fibroblástico-2 (FGF-2) e fator de crescimento transformador-beta1 (TGF-β1). Resultados: As diferentes centrifugações geraram perfis sistematicamente diferentes referentes ao número de plaquetas e de leucócitos. O sistema da Medtronic originou produto com a maior concentração de plaquetas, e o método aberto com a menor concentração de plaquetas. Os resultados da análise de citocinas demonstraram que os diferentes tipos de centrifugação originaram produtos com elevadas concentrações de interleucina 8 e interleucina 1β. O sistema aberto resultou em produto com elevados níveis de interleucina 6. As demais citocinas e quimiocinas mensuradas foram similares entre os sistemas. O produto obtido com o método aberto apresentou níveis superiores de TGF-β1 em relação aos demais sistemas e reduzidos níveis de FGF-2. Conclusão: Os elementos figurados, fatores de crescimento e citocinas, em amostras de plasma rico em plaquetas, variaram conforme a técnica de centrifugação utilizada.

Citotoxicidade do Viscum album L. em células de carcinoma epidermoide da cavidade bucal / Cytotoxicity of mistletoe (Viscum album L. ) in oral squamous cell carcinoma

O carcinoma epidermoide de cabeça e pescoço é uma doença bastante complexa com diversos fatores etiológicos, além de mudanças distintas das estruturas e bases moleculares as quais provocam determinados eventos que culminam no surgimento desses tumores. Globalmente, também é classificada como uma das doenças mais comuns desta região. Principalmente nos países europeus os extratos de Viscum album L. (VA) (mistletoe) vêm sendo utilizado como terapia coadjuvante, com resultados bastante promissores em diversos tipos de tumores malignos. Estudos in vitro demonstraram que vários tipos de VA são capazes de provocar efeitos citotóxicos em células de carcinoma, ativando a cascata apoptótica ou levando as células à necrose. Este estudo teve como objetivo verificar a ação de três tipos de extratos de VA, não estudados anteriormente: Iscador Qu Spezial, Iscador P e Iscador M, em células de linhagem de carcinoma epidermoide de língua (SCC9 e SCC25). A concentração de 0.3 mg/mL (IC50) dos fármacos foi capaz de levar essas células à apoptose. Foi concluído que os extratos de VA possuem efeito citotóxico nas células de carcinoma epidermoide de língua (SCC9 e SCC25), entretanto as SCC9 possuem maior resistência à ação dos fármacos. Iscador Qu Spezial e Iscador M possuem maior potencial citotóxico em ambas as células, quando comparado ao Iscador P.(AU)

Head and neck squamous cell carcinoma is a complex disease with several etiologic factors and different molecular changes that may trigger certain events; it is also globally one of the most common malignancies in this topography. Extracts from Viscum album L. (VA) (mistletoe) have been used as adjuvant therapies with promising results in several types of cancer, mainly in European countries. In vitro studies have demonstrated that various types of VA may have cytotoxicity in carcinoma cells, activating the apoptotic cascade or leading cells to necrosis. This study aimed to verify the effect of three types of VA extracts (Iscador Qu Spezial, Iscador P and Iscador M) in cell lines of squamous cell carcinoma of the tongue (SCC9 and SCC25), not previously studied. A concentration of 0.3 mg/mL (IC50) of the drugs was capable to induce apoptosis. It was concluded that VA extracts have a cytotoxic effect on SCC9 and SCC25 cell lines, but while SCC9 cell line was more resistant to the action of the drugs. Iscador Qu Spezial and Iscador M have higher cytotoxic potential in both cell lines compared to Iscador P.(AU)

Salivary transforming growth factor alpha in patients with Sjögren's syndrome and reflux laryngitis.

INTRODUCTION: Saliva plays a key role in the homeostasis of the digestive tract, through its inorganic components and its protein growth factors. Sjögren's syndrome patients have a higher prevalence of gastroesophageal reflux disease and laryngopharyngeal reflux. Decreased salivary transforming growth factor alpha levels were observed in dyspeptic patients, but there have been no studies in patients with Sjögren's syndrome and laryngopharyngeal reflux. OBJECTIVE: To compare the salivary transforming growth factor alpha levels of patients with Sjögren's syndrome and laryngopharyngeal reflux to those of healthy controls. METHODS: This is a prospective controlled study. Twelve patients with Sjögren's syndrome and laryngopharyngeal reflux and 11 controls were prospectively evaluated. Spontaneous and stimulated saliva samples were obtained to establish salivary transforming growth factor alpha concentrations. RESULTS: The salivary transforming growth factor alpha levels of patients were significantly higher than those of healthy controls. Five patients with laryngopharyngeal reflux also had erosive esophagitis; their salivary transforming growth factor alpha levels were comparable to controls. CONCLUSION: Salivary transforming growth factor alpha level was significantly higher in patients with Sjögren's syndrome and laryngopharyngeal reflux when compared to the control group.

Salivary transforming growth factor alpha in patients with Sjögren's syndrome and reflux laryngitis / Estudo da concentração salivar do fator transformador de crescimento alfa em indivíduos com Sindrome de Sjögren e refluxo laringofaríngeo

Introduction: Saliva plays a key role in the homeostasis of the digestive tract, through its inorganic components and its protein growth factors. Sjögren's syndrome patients have a higher prevalence of gastroesophageal reflux disease and laryngopharyngeal reflux. Decreased salivary transforming growth factor alpha levels were observed in dyspeptic patients, but there have been no studies in patients with Sjögren's syndrome and laryngopharyngeal reflux. Objective: To compare the salivary transforming growth factor alpha levels of patients with Sjögren's syndrome and laryngopharyngeal reflux to those of healthy controls. Methods: This is a prospective controlled study. Twelve patients with Sjögren's syndrome and laryngopharyngeal reflux and 11 controls were prospectively evaluated. Spontaneous and stimulated saliva samples were obtained to establish salivary transforming growth factor alpha concentrations. Results: The salivary transforming growth factor alpha levels of patients were significantly higher than those of healthy controls. Five patients with laryngopharyngeal reflux also had erosive esophagitis; their salivary transforming growth factor alpha levels were comparable to controls. Conclusion: Salivary transforming growth factor alpha level was significantly higher in patients with Sjögren's syndrome and laryngopharyngeal reflux when compared to the control group. .

Introdução: A saliva exerce influência primordial na homeostase do sistema digestório, pelos seus componentes inorgânicos e pelos fatores de crescimento. Indivíduos com sindrome de Sjögren (SS) apresentam maior incidência da doença do refluxo gastroesofágico (DRGE) e do refluxo laringofaríngeo (RLF). Concentrações salivares diminuídas do fator transformador de crescimento alfa (TGF-α) foram observadas em doentes dispépticos, porém não há estudos em populações com SS e RLF. Objetivo: Comparar concentrações salivares do TGF-α; de indivíduos com SS e RLF a de controles saudáveis. Método: Trata-se de um estudo prospectivo controlado. Doze pacientes com SS e RLF e 11 indivíduos controles saudáveis tiveram amostras salivares espontâneas e estimuladas coletadas para estabelecer concentração de TGF-α. Resultados: A concentração salivar de TGF-α; foi estatisticamente maior no grupo estudo para ambas amostras. Este aumento foi confirmado nos sete indivíduos do grupo estudo que não apresentavam esofagite erosiva quando comparados ao grupo controle, porém não houve diferença estatística da concentração de TGF-α; entre pacientes do grupo estudo que apresentava mesofagite erosiva em comparação ao grupo controle. Conclusão: A concentração salivar de TGF-α; foi estatisticamente maior no grupo de indivíduos com SS e RLF, sem esofagite erosiva. .