Effects of TYROBP Deficiency on Neuroinflammation of a Alzheimer's Disease Mouse Model Carrying a PSEN1 p.G378E Mutation / 中国医学科学杂志(英文版)

Objective To study the effects of TYRO protein kinase-binding protein (TYROBP) deficiency on learning behavior, glia activation and pro-inflammatory cycokines, and Tau phosphorylation of a new Alzheimer's disease (AD) mouse model carrying a PSEN1 p.G378E mutation.Methods A new AD mouse model carrying PSEN1 p.G378E mutation was built based on our previously found AD family which might be ascribed to the PSEN1 mutation, and then crossed with TYROBP deficient mice to produce the heterozygous hybrid mice (PSEN1G378E/WT; Tyrobp+/-) and the homozygous hybrid mice (PSEN1G378E/G378E; Tyrobp-/-). Water maze test was used to detect spatial learning and memory ability of mice. After the mice were sacrificed, the hippocampus was excised for further analysis. Immunofluorescence was used to identify the cell that expresses TYROBP and the number of microglia and astrocyte. Western blot was used to detect the expression levels of Tau and phosphorylated Tau (p-Tau), and ELISA to measure the levels of pro-inflammatory cytokines. Results Our results showed that TYROBP specifically expressed in the microglia of mouse hippocampus. Absence of TYROBP in PSEN1G378E mutation mouse model prevented the deterioration of learning behavior, decreased the numbers of microglia and astrocytes, and the levels of interleukin-6, interleukin-1β and tumor necrosis factor-α in the hippocampus (all P < 0.05). The ratios of AT8/Tau5, PHF1/Tau5, pT181/Tau5, pT231/Tau5 and p-ERK/ERK were all higher in homozygous hybrid mice (PSEN1G378E/G378E; Tyrobp-/- mice) compared with PSEN1G378E/G378E mice (all P < 0.05). Conclusions TYROBP deficiency might play a protective role in the modulation of neuroinflammation of AD. However, the relationship between neuroinflammation processes involving microglia and astrocyte activation, and release of pro-inflammatory cytokines, and p-Tau pathology needs further study.

Colecalciferol regula os parâmetros hematológicos e a produção de citocinas pró-inflamatórias renais em camundongos diabéticos e nas células RAW 264. 7 / Colecalciferol regulates haematological parameters and the production of renal proinflammatory cytokines in diabetic mice and RAW 264. 7 cells

Os efeitos causados pelo tratamento em conjunto da insulina e do colecalciferol em indivíduos diabéticos não estão completamente elucidados. O presente trabalho avaliou o efeito de ambos os hormônios nos rins, no fígado, no coração e nos parâmetros hematológicos de camundongos machos (C57BL/6) sadios e diabéticos, bem como a ação do colecalciferol (in vitro) na resposta imunológica desenvolvida pelas células RAW 264.7 e pelos macrófagos peritoneais (MP) após estímulo com lipopolissacarídeo (LPS). Após dez dias da administração da aloxana (60 mg/kg), animais diabéticos exibiram redução do ganho de peso corporal e hiperglicemia quando comparados aos animais que receberam salina. No sétimo dia do período experimental, foi verificado que animais diabéticos que não receberam nenhum hormônio, em relação aos não diabéticos, exibiram redução do peso corporal, dos níveis de hemoglobina (Hb), hematócrito, hematimetria, insulina, TNF-α e IL-6 (coração) e aumento da glicemia, da relação peso corpóreo/peso rim esquerdo, das concentrações séricas de ureia, creatinina, Fosfatase Alcalina (FAL), Lactato desidrogenase (LDH) e lactato, fator de necrose tumoral (TNF)-α, interleucina (IL)-6 e IL-10 (no rim); o tratamento com insulina (1 UI/300 mg/dL glicemia), em relação aos animais diabéticos não tratados, promoveu aumento do peso corporal, das concentrações séricas de insulina e redução da glicemia, das concentrações séricas de ureia e da razão TNF-α/IL-10 (coração); o tratamento com colecalciferol (800 UI/dia), em relação aos animais diabéticos não tratados, promoveu aumento das concentrações séricas de 25-hidroxicolecalciferol [25(OH)D], Hb, hematócrito, hematimetria, IL-10 (coração) e reduziu IL-6, IL-10, TNF-α e EPO (rim); os animais diabéticos tratados com insulina, em relação aos animais diabéticos suplementados com colecalciferol apresentaram aumento do peso corpóreo, de ureia sérica, IL-6 e TNF-α (coração) e redução da glicemia, das concentrações séricas de lactato, de IL-6, TNF-α, IL-10 e EPO (rim); os animais -que receberam ambos os hormônios, em relação aos animais tratados com insulina, apresentaram aumento sérico de insulina e lactato; os animais diabéticos que receberam ambos os hormônios, em relação aos animais diabéticos tratados com colecalciferol, exibiram aumento sérico de 25(OH)D, de insulina, além da redução das concentrações de IL-10, da razão de TNF-α/IL-10 e TNF-α/IL-6 (coração); animais diabéticos que receberam ambos os hormônios, em relação aos diabéticos não suplementados com colecalciferol, exibiram: aumento de insulina sérica e redução das concentrações séricas de ureia e das razões renal e hepática de TNF-α/IL-6; células RAW 264.7 estimuladas pelo LPS e tratadas com 100 nM colecalciferol exibiram maior expressão da CYP27B1 e redução na liberação de mediadores inflamatórios quando comparadas ao grupo estimulado pelo LPS. Entretanto, não foi observado o mesmo efeito nos MP. Em conjunto, os resultados sugerem que: 1) em animais diabéticos, o colecalciferol pode modular parâmetros hematológicos e que a insulina pode melhorar a função renal, bem como a recuperação do peso corporal; 2) o colecalciferol pode ser metabolizado pelas células RAW 264.7 e modular a resposta imunológica desencadeada pelo LPS

The effects caused by the treatment of insulin and cholecalciferol in diabetic subjects are not completely elucidated. The present study evaluated the effect of both hormones on the kidneys, liver, heart and hematological parameters of healthy and diabetic male mice (C57BL/6), as well as the action of cholecalciferol (in vitro) on the immune response developed by the cells RAW 264.7 and peritoneal macrophages (MP) after stimulation with lipopolysaccharide (LPS). After ten days of alloxan administration (60 mg/kg), diabetic animals exhibited a reduction in body weight gain and hyperglycemia when compared to animals that received saline. On the seventh day of the experimental period, it was verified that diabetic animals that did not receive any hormones, in relation to non-diabetics, showed reduction of body weight, hemoglobin (Hb), hematocrit, hematimetry, insulin, TNF-α and IL- 6 (heart) and increased glycemia, body weight / left kidney weight, serum urea, creatinine, Phosphatase Alkaline, lactate dehydrogenase (LDH) and lactate levels, tumor necrosis factor (TNF) interleukin (IL) -6 and IL-10 (in the kidney); diabetic mice treated with insulin (1 IU / 300 mg/dL glycemia) in relation to untreated diabetic animals promoted increased body weight, serum insulin levels and blood glucose lowering, serum urea levels and TNF-α ratio / IL-10 (heart); diabetic animals treated with cholecalciferol (800 IU/day), in relation to untreated diabetic animals, exhibited increased serum levels of 25-hydroxycholecalciferol [25 (OH) D], Hb, hematocrit, hematimetry, IL-10 (heart) and reduced IL-6, IL-10, TNF-α and EPO (kidney);insulin-treated diabetic animals compared to diabetic animals supplemented with cholecalciferol exhibited an increase of body weight, serum urea, IL-6 and TNF-α (heart) and a reduction of glycaemia, serum lactate levels, IL-6, TNF- α, IL-10 and EPO (kidney); animals that received both hormones, compared to animals treated with insulin exhibited an increase of insulin and lactate serum levels; diabetic animals that received both hormones, compared to diabetic animals treated with cholecalciferol, exhibited an increase of 25(OH)D and insulin serum levels, and a reduction of IL-10, TNF-α/IL-10 and TNF-α/IL-6 ratios (heart); diabetic animals that received both hormones, compared to diabetic animals not supplemented with cholecalciferol, exhibited an increase of insulin and reduced urea serum levels and reduced renal and hepatic TNF-α/IL-6 ratios; LPS-stimulated RAW 264.7 cells and treated with 100 nM cholecalciferol exhibited greater CYP27B1 expression and reduced release of inflammatory mediators when compared to the LPS-stimulated group. However, the same effect was not observed in PM. Taken together, the results suggest that: 1) in diabetic animals, cholecalciferol may modulate hematological parameters and that insulin may improve renal function as well as recovery of body weight; 2) cholecalciferol can be metabolized by RAW 264.7 cells and modulate the immune response triggered by LPS

Influência da insulina sobre vias de sinalização envolvidas na peritonite provocada por inoculação de staphylococcus aureus em animais sadios e diabéticos / Influence of insulin on signaling pathways involved in peritonitis caused by inoculation of staphylococcus aureus in healthy and diabetic animals

Dentre tantas complicações do diabetes mellitus (DM), a infecção por bactérias comuns da microbiota superficial da pele como, por exemplo, a bactéria Gram-positiva Staphylococcus aureus, causadora de infecções como a peritonite, com altos índices de hospitalização e morte. A hipótese deste trabalho é a que o efeito da insulina na ativação das vias de sinalização MAPK, PKC e PI3K em peritonite induzida por S. aureus em animais diabéticos e não diabéticos possa regular a produção de citocinas. Foram utilizadas amostras de fígado, rim, linfonodos peritoniais e baço de animais oriundos de estudo anterior (Projeto FCF/USP-375), no qual animais diabéticos (aloxana, 42 mg/kg, i.v., 10 dias) e não-diabéticos com peritonite decorrente da infecção por S. aureus receberam uma dose de 4 UI e 1 UI de insulina NPH, respectivamente, por via subcutânea, 2 horas antes da infecção com S. aureus, e outras 3 doses de 2 UI e 0,5 UI às 17h00', respectivamente. A glicemia foi determinada no dia anterior, 10 dias após a injeção de aloxana e após os tratamentos com insulina. Em amostras de fígado, rim, linfonodo e baço dos animais supra citados foram avaliados a dosagem de citocinas (IL-1ß, IL-4, IL-10, TNF-α, CINC-1, CINC-2 e CINC-3) por ensaios de enzima-imunoensaio (ELISA); em homogenato de fígado foram avaliadas a expressão das moléculas das vias MAPK (fosfo P-38, fosfo ERK p42/44), PKC (fosfo PKC-α, fosfo PKC-δ) e PI3K (fosfo-AKT) pelo método de Western Blotting. Na avaliação do fígado, a insulina foi capaz de aumentar a concentração das citocinas IL-4 e TNF-α que apresentavam-se diminuídas em animais não diabéticos, em relação aos animais não diabéticos e não infectados, mas nos animais diabéticos, na infecção pela cepa N315, a insulina diminuiu a concentração de IL-4, que não estava alterada pela infecção, e não foi capaz de aumentar a concentração de IL-1ß que estava diminuída na infecção, em relação aos animais diabéticos e não infectados. Em linfonodos peritoniais de animais não diabéticos infectados pela cepa N315, a insulina diminuiu a produção de IL-1ß e IL-10, que não estavam alteradas na infecção, e diminuiu a concentração de IL-4, que estava aumentada na infecção, em relação aos animais não diabéticos e não infectados; em animais diabéticos, a insulina diminuiu a produção das citocinas IL-1ß e CINC-1, que estavam aumentadas, e aumentou a concentração de IL-10, que estava diminuída na infecção com a cepa N315, mas baixou a concentração de IL-4, em relação aos animais infectados, e na infecção pela cepa ATCC, a insulina aumentou a produção de IL-1ß, CINC-1 e CINC-3 dos animais tratados, em relação aos infectados e não tratados. Em baço, a insulina diminuiu a produção de IL-10 na infecção pela cepa ATCC tanto em animais não diabéticos quanto em animais diabéticos e, nesse último grupo, também aumentou a produção de CINC-3 em relação aos animais diabéticos não infectados; na infecção com a cepa N315, a insulina não aumentou a concentração de IL-1ß e TNF-α, que estavam diminuídas na infecção. Em rim, não houveram alterações significativas na produção de citocinas na infecção com nenhuma das cepas estudadas, nem para os grupos diabéticos, nem para os não diabéticos. Verificou-se que os animais diabéticos apresentam maior alteração tanto nas vias de sinalização estudadas quando na produção de citocinas pró-inflamatórias, quando comparados aos animais não diabéticos, na infecção por ambas as cepas de S. aureus estudadas. Assim, os resultados obtidos sugerem que o tratamento com insulina possa modular parcialmente a produção das citocinas IL-1ß, TNF-α e IL-10 no fígado e nos linfonodos peritoniais dos animais infectados principalmente pela cepa N315 de S.aureus, modulando parcialmente a expressão das moléculas da via de sinalização (MAPK e PKC), envolvidas na produção dessas citocinas

Among so many complications of diabetes mellitus (DM), infection by common bacteria of the superficial microbiota of the skin, for example, a gram-positive bacteria Staphylococcus aureus, causing infections like peritonitis, with high rates of hospitalization and death. The hypothesis of this study is that the effect of insulin on the activation of MAPK, PKC and PI3K signaling pathways in peritonitis induced by S. aureus in diabetic and non-diabetic animals may regulate the production of proinflammatory cytokines. Liver, kidney, peritonial lymph nodes and spleen samples of animals from the previous study (FCF / USP-375 Project) were used in this project; diabetic animals (alloxan, 42 mg / kg, iv, 10 days) and non-diabetic animals with peritonitis due to S. aureus infection received one dose of 4 IU and 1 IU of NPH insulin, respectively, subcutaneously, 2 hours before infection with S. aureus, and another 3 doses of 2 IU and 0.5 IU at 5:00 p.m., respectively. Blood glucose was determined the day before, 10 days after alloxan injection and after insulin treatments. In the liver, kidney, lymph node and spleen samples of the above-mentioned animals the cytokines (IL-1ß, IL-4, IL-10, TNF-α, CINC- 2 and CINC-3) by enzyme-immunoassay (ELISA) assays; we avaliated, by Western Blotting, the signaling pathways MAPK (phospho-P-38, phospho ERK p42 / 44), PKC (phospho PKC-α and phospho PKC-δ) and PI3K (phospho AKT) in liver, insulin was able to increase the concentration of cytokines IL-4 and TNF-α that were decreased in non-diabetic animals, in relation to non-diabetic and non-infected animals, but in diabetic animals, in strain N315, insulin decreased the concentration of IL-4, which was not altered by the infection, and was not able to increase the concentration of IL-1ß that was decreased in infection, relative to diabetic and uninfected animals. In peritonial lymph nodes from non-diabetic animals infected with the N315 strain, insulin decreased the production of IL-1ß and IL-10, which were not altered in the infection, and decreased the concentration of IL-4, which was increased in infection, in relation to non-diabetic and non-infected animals; in diabetic animals, insulin decreased IL-1ß and CINC-1 which were increased, and increased the concentration of IL-10, which was decreased in infection with strain N315, but decreased the concentration of IL-4 in Infected animals, and in infection by the ATCC strain, insulin increased the production of IL-1ß, CINC-1 and CINC-3 of treated animals over infected and untreated animals. In spleen, insulin decreased IL-10 production on infection by the ATCC strain in both non-diabetic and diabetic animals and, in this last group, also increased the production of CINC-3 in relation to uninfected diabetic animals; in infection with the N315 strain, insulin did not increased the concentration of IL-1ß and TNF-α, which were decreased in infection. In kidney, there were no significant changes in cytokine production in infection with any of the strains studied, neither for diabetic groups nor for non-diabetics. It was verified that diabetic animals present a greater alteration both in the signaling pathways studied and in the production of pro-inflammatory cytokines, when compared to non-diabetic animals, in the infection by both strains of S. aureus studied. Thus, the results suggest that insulin treatment may partially modulate the production of IL-1ß, TNF-α and IL-10 cytokines in the liver and in the peritonial lymph nodes of animals infected mainly with S. aureus strain N315, since they partially modulating the expression of signaling pathway molecules (MAPK and PKC), involved in the production of these cytokines

Papel da insulina na vigência da peritonite decorrente da infecção por Staphylococcus aureus em animais diabéticos e sadios / Insulin role in peritonitis presence due to Staphylococcus aureus infection in diabetic and healthy animals

A nefropatia diabética é uma doença crônica caracterizada por falência renal, que torna necessária a hemodiálise. A diálise peritoneal é uma alternativa para a hemodiálise, porém causa peritonite e morte, principalmente devido à infecção com Staphylococcus aureus, especialmente em pacientes imunodeprimidos, como pacientes diabéticos. Nossa hipótese é que a insulina possa modular a peritonite causada por S. aureus. Para tanto, investigamos sua intervenção, após a indução de diabetes mellitus, na infecção peritoneal por cepas diferentes de S. aureus, analisando os mecanismos moleculares (produção/liberação de citocinas, expressão de moléculas de adesão) e a atividade microbicida dos macrófagos peritoneais envolvidos. Ratos Wistar, machos, diabéticos (aloxana, 42 mg/kg, i.v., 10 dias) e respectivos controles (salina, i.v.) foram submetidos à injeção intraperitoneal de uma suspensão de S. aureus (5x109 CFU/mL) ou volume equivalente de PBS estéril. Os animais foram submetidos a dois tratamentos com insulina NPH: 1) dose única (1UI e 4UI respectivamente, controle e diabético), administrada por via subcutânea; ou, 2) com 4 doses sendo a primeira administrada 2 horas antes da infecção, seguida de metade desta dose às 17 horas e no mesmo horário pelos próximos 2 dias (dose inicial 4UI e 1UI, grupo diabético e grupo controle, respectivamente), passadas 16 horas da última dose de insulina, a glicemia foi determinada e, em seguida, foi realizada eutanásia e coleta de amostras. Avaliamos: a) número de células no lavado peritoneal (LPe), leucograma e glicemia (monitor de glicose); b) níveis séricos de corticosterona e insulina (ELISA); c) concentrações de citocinas (IL-1ß, TNF-α, IL-6, IFN-γ, IL-4, IL-10, IL-12) e quimiocinas (CINC-1, CINC-2, CINC-3) no sobrenadante do LPe (ELISA); d) expressão de moléculas de adesão (P-selectina, PECAM-1, ICAM-1) no endotélio vascular (imunoistoquímica); e) atividade microbicida. Após a infecção com a cepa ATCC 25923, comparados aos não infectados, ratos diabéticos apresentaram aumento no número de leucócitos (350%) e nas concentrações de CINC-1 (1900%), IL-1ß (1300%), IFN-γ (280%), IL-4 (800%). O tratamento destes animais com dose única de insulina diminuiu as concentrações de CINC-1 (17%) e IFN-γ (30%) e o leucócitos (55%); e aumentou a concentração de IL-4 (260%); enquanto o tratamento com 4 doses diminuiu o número de leucócitos (82%) e as concentrações de CINC-1 (96%) e CINC-2 (45%); e, aumentou as concentrações de TNF-α (270%), IFN-γ (220%), IL-1ß (42%), IL-6 (760%) e a expressão de ICAM-1 (1360%); enquanto as concentrações de CINC-3, IL-10 e IL-12 não foram alteradas pelos tratamentos com insulina. Após a infecção com a cepa N315 HLA+, comparados aos não infectados, ratos diabéticos apresentaram aumento do número de leucócitos (200%), nas concentrações de CINC-1 (1000%), IL-4 (860%), IFN-γ (200%) e na expressão de PECAM-1 (800%) e diminuição de CINC-2 (92%). O tratamento destes animais com dose única de insulina diminuiu a concentração de CINC-1 (85%); e aumentou a concentração de CINC-2 (2030%), IL-1ß (370%) e IL-4 (250%); enquanto o tratamento com 4 doses diminuiu a concentração de CINC-1 (92%); e, aumentou número de leucócitos (66%), as concentrações de CINC-2 (100%), IL-1ß (490%), IL-6 (1870%) e IFN-γ (330%), e os outros parâmetros não foram modificados pelos diferentes tratamentos com insulina. Estes dados sugerem que a insulina possa modular a peritonite induzida por cepas diferentes de S. aureus, controlando pelo menos em parte, o infiltrado inflamatório, a produção das citocinas CINC-1, CINC-2, IL-4, IL-6, IFN-γ, TNF-α e IL-1ß; e, consequentemente a expressão P-selectina e PECAM-1 no endotélio vascular do mesentério

Diabetic nephropathy is a chronic disease characterized by kidney failure, so hemodialysis is necessary. Peritoneal dialysis is an alternative to hemodialysis, but causes peritonitis and death primarily due to infection by Staphylococcus aureus, especially in immunocompromised patients, such as diabetics. Our hypothesis is that insulin can modulate peritonitis caused by S. aureus, therefore, we investigated its action, after diabetes induction, in peritoneal infection with different S. aureus strains, analyzing the molecular mechanisms (cytokines production/release, adhesion molecules expression) and the microbicidal activity of peritoneal macrophages involved. Wistar male diabetic (alloxan, 42 mg/kg, iv, 10 days) and their respective controls (saline,iv) were subjected to intraperitoneal injection of S. aureus suspension (5x109CFU/mL) or an equivalent volume of PBS sterile. Animals were submitted to two treatments with NPH insulin administered subcutaneously : single dose (1IU and 4IU respectively , control and diabetic) 8 hours prior to euthanasia ; or 4 doses : first dose 2 hours before infection (4IU and 1IU , diabetic and control group, respectively), then half this dose of 17 pm and in the same time for the next 2 days, after 16 hours of the last dose, blood glucose was determined, and then it was carried out euthanasia and sampling. We evaluated: a) number of cells in peritoneal wash (PW), white blood cell count and blood sugar (glucose monitor) ; b) serum insulin and corticosterone (ELISA); c) concentrations of cytokines (IL-1ß, TNF-α, IL-6, IFN-γ, IL-4, IL-10, IL-12) and chemokines (CINC -1, CINC-2, CINC-3 ) in supernatant of the SBA assay (ELISA); d) expression of adhesion molecules (P- selectin, ICAM-1, PECAM-1) in vascular endothelium ( immunohistochemistry); e) microbicidal activity. After infection with ATCC 25923 strain, compared to uninfected, diabetic rats showed an increase in leukocytes number (350%) and in concentrations of CINC-1 (1900%), IL-1ß (1300%), IFN-γ (280%), IL-4 (800%). Treatment of these animals with a single dose of insulin decreased concentrations of CINC-1 (17%) and IFN-γ (30%) and leukocytes number (55%); and increased IL-4 concentrations (260%); while treatment with 4 doses decreased leukocytes number (82%) and concentrations of CINC-1 (96%) and CINC-2 (45%); and increased concentrations of TNF-α (270%), IFN-γ (220%), IL-1ß (42%), IL-6 (760%) and expression of ICAM-1 (1360%); while the concentrations of CINC-3, IL-10 and IL-12 were not affected by treatments with insulin. After infection with N315 HLA+ strain, compared to uninfected, diabetic rats showed an increase in leukocytes (200%), concentrations of CINC-1 (1000%), IL-4 (860%), IFN-γ (200%) and in the expression of PECAM-1 (800%); and CINC-2 decrease (92%). Treatment of these animals with a single dose of insulin decreased the concentration of CINC-1 (85%); and increased concentrations of CINC-2 (2030%), IL-1ß (370%) and IL-4 (250%); while treatment with 4 doses decreased the concentration of CINC-1 (92%); and increased leukocytes number (66%), concentrations of CINC-2 (100%), IL-1ß (490%), IL-6 (1870%) and IFN-γ (330%), and other parameters were not modified by treatments with insulin. These results suggest that both S. aureus strains activate differently the innate response during peritonitis, and insulin was not always able to modulate this response

Biologic Response of Degenerative Living Human Nucleus Pulposus Cells to Treatment with Cytokines

PURPOSE: To investigate the molecular responses of various genes and proteins related to disc degeneration upon treatment with cytokines that affect disc-cell proliferation and phenotype in living human intervertebral discs (IVDs). Responsiveness to these cytokines according to the degree of disc degeneration was also evaluated. MATERIALS AND METHODS: The disc specimens were classified into two groups: group 1 (6 patients) showed mild degeneration of IVDs and group 2 (6 patients) exhibited severe degeneration of IVDs. Gene expression was analyzed after treatment with four cytokines: recombinant human bone morphogenic protein (rhBMP-2), transforming growth factor-beta (TGF-beta), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha). Molecular responses were assessed after exposure of cells from the IVD specimens to these cytokines via real-time polymerase chain reaction and immunofluorescence staining. RESULTS: mRNA gene expression was significantly greater for aggrecan, type I collagen, type II collagen, alkaline phosphatase, osteocalcin, and Sox9 in group 1 than mRNA gene expression in group 2, when the samples were not treated with cytokines. Analysis of mRNA levels for these molecules after morphogen treatment revealed significant increases in both groups, which were much higher in group 1 than in group 2. The average number of IVD cells that were immunofluorescence stained positive for alkaline phosphatase increased after treatment with rhBMP-2 and TGF-beta in group 1. CONCLUSION: The biologic responsiveness to treatment of rhBMP-2, TGF-beta, TNF-alpha, and IL-1beta in the degenerative living human IVD can be different according to the degree of degeneration of the IVD.

In vitro assessment of cytokines interactions with Balamuthia mandrillaris using human brain microvascular endothelial cells

Balamuthia amoebic encephalitis [BAE] is a life threatening human disease which, always lead to death. Amoebae invasion of the bloodstream is considered an important step in BAE followed by their haematogenous spread. It is more likely that Balamuthia mandrillaris enters into the central nervous system through blood-brain barrier [BBB] sites. The objective of the present study was to determine the impact of cytokines on biological properties of alamuthia in vitro. Human brain microvascular endothelial cells [HBMEC], which constitutes the BBB were used in vitro test model for the present investigation. It was observed that Balamuthia exhibited >90% binding and >70% cytotoxicity to HBMEC. However, cytokines did not affect amoebic binding and cytotoxicity except lipopolysaccharide [LPS] which reduced Balamuthia-mediated HBMEC cytotoxicity. It is also important to note that amoebic numbers were reduced in the presence of LPS within 24 h. We have shown previously the bacterial uptake by Balamuthia is very limited which is further investigated in the presence of cytokines and observed a slight reduction of bacterial uptake during phagocytosis assay. Zymography assays revealed there is no effect of cytokines on proteolytic activity of Balamuthia. Overall we described for the first time that cytokines has no inhibitory effects on biological properties of Balamuthia in vitro.

Avaliação do status de magnésio e da sua associação com o estresse oxidativo e as citocinas inflamatórias na pré-eclâmpsia / Assessment of the magnesium status and its association with oxidative stress and inflammatory cytokines in preeclampsia

O objetivo deste trabalho foi avaliar o status de magnésio (Mg) e a sua relação com o estresse oxidativo e as citocinas inflamatórias na pré-eclâmpsia (PE). Participaram do estudo, 18 gestantes saudáveis (controle - CT) e 18 gestantes com PE, diagnosticadas com pressão arterial >= 140/90 mmHg, proteinúria >= 0,3 g/24 h e sem doenças associadas. Sangue e urina de 24 horas foram coletados para análise de status de Mg, estresse oxidativo [malondialdeído (MDA), 8-isoprostano urinário e a atividade antioxidante das enzimas catalase (CAT) e glutationa peroxidase (GSH-Px)], a concentração de óxido nítrico (NO), e das citocinas inflamatórias [proteína C reativa, interleucina 6 (IL-6) e fator de necrose tumoral (TNF-α)]; Foi aplicado um questionário quantitativo de frequência alimentar para gestantes. As comparações entre os grupos foram feitas pelos testes Qui-quadrado, t-Student ou Mann Whitney. O coeficiente de correlação de Spearman foi usado para verificar associação entre as variáveis. A análise do Receiver Operating Characteristic (ROC) foi realizada para identificar as variáveis que melhor discriminassem os grupos (α=5%). As concentrações de Mg plasmático e eritrocitário, bem como a concentração de NO, a atividade da CAT e as concentrações de TNF-α e IL-6 foram maiores na PE do que no CT. Associações positivas entre o Mg plasmático e a proteinúria (p=0,04), o TNFα (p=0,03) e a IL-6 (p=0,02) foram verificadas; associações negativas foram encontradas entre a atividade da CAT e a concentração de 8-isoprostano urinário (p=0,02) e entre a atividade da GSH-Px e os níveis de pressão arterial diastólica (p=0,01). A análise ROC mostrou que o Mg plasmático e o TNF-α foram as variáveis que mellhor discriminaram as gestantes com PE das CT. Os resultados mostraram que o estresse oxidativo não foi evidente na fisiopatologia da PE, possivelmente devido aos mecanismos antioxidantes compensatórios do organismo. A inflamação e os eventos inerentes à PE, como vasoconstrição, podem ter promovido as alterações no status de Mg

The aim of this study was to assess the magnesium (Mg) status and its relationship with oxidative stress and inflammatory cytokines in preeclampsia (PE). Were included 18 healthy pregnant women (CT- control) and 18 PE, diagnosed with blood pressure >= 140/90 mmHg, proteinuria >= 0,3 g/24 h, and without other diseases. Blood and 24h urine were collected for analyses of the Mg status, oxidative stress [malondialdehyde (MDA), 8-isoprostane urinary and activities of the antioxidant enzymes: catalase (CAT) and glutathione peroxidase (GSH-Px)], nitric oxide (NO) and inflammatory cytokines concentrations [protein C reactive, interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α); Furthermore, a quantitative food frequency questionnaire was applied to pregnant women. The comparisons between groups were done by Chi-square, t-Student or Mann Whitney tests. Spearman correlation coefficient was used to verify association among variables and the Receiver Operating Characteristic (ROC) analysis was performed to identify variables that better discriminated the groups (α = 5 %). The Mg concentration, in plasma and in erythrocyte, as well as NO concentration, CAT activity and TNF-α and IL-6 concentrations were higher in PE than CT group. Positive associations between plasma Mg and proteinuria (p=0,04), TNF-α (p=0,03) and IL-6 (p=0,02) were verified; Negative associations were found between CAT activity and 8-isoprostane urinary concentration (p=0,02) and between GSH-Px activity and diastolic blood pressure levels (p=0,01). ROC analyses showed that plasma Mg and TNF-α were the variables which better discriminate pregnant women with PE from CT. The results showed that oxidative stress was not evident in physiopathology of PE, possibly due to compensatory antioxidant mechanisms present in the body. The inflammatory and the events inherent to PE, such as vasoconstriction, possibly have promoted changes in Mg status

Avaliação do efeito preemptivo da s(+)-cetamina por via peridural para histerectomia: concentrações plasmáticas de interleucinas / Evaluation of preemptive effect of epidural s(+)-ketamine for hysterectomy: plasmatic concentrations of interleukins

JUSTIFICATIVA E OBJETIVOS: Alguns estudos demonstraram que a cetamina inibe a produção de citocinas. O objetivo deste estudo foi avaliar o efeito analgésico preemptivo e citocinas plasmáticas (IL-6, TNF-α e IL-10) de S(+)-cetamina por via peridural em histerectomia. MÉTODO: Foi realizado estudo duplo-encoberto em 29 pacientes. Pacientes do Grupo 1 receberam 13 mL de bupivacaína a 0,25 por cento com 25 mg de S(+)-cetamina 30 minutos antes da incisão cirúrgica, e 15 mL de solução salina fisiológica, 30 minutos após, por via peridural. Pacientes do Grupo 2 receberam 15 mL de salina 30 minutos antes da incisão cirúrgica, seguido por 13 mL de bupivacaína 0,25 por cento, mais 25 mg de S (+)-cetamina 30 minutos após. A analgesia pós-operatória foi feita com bupivacaína e fentanil por via peridural. Quando necessário, foi utilizado 1 g de dipirona. Foram avaliados: concentração de citocinas, intensidade da dor, o tempo da primeira solicitação de analgésico e a quantidade total de analgésico utilizado. RESULTADOS: O tempo para a primeira solicitação de analgésico foi de 61,5 minutos no Grupo 1 e 69,0 no Grupo 2, sem diferença entre os grupos. Não houve diferença entre os grupos para a dose total de fentanil usada no Grupo 1 (221,4 µg) e Grupo 2 (223,3 µg). Foi obtido efeito analgésico semelhante nos grupos, exceto em T12 (Grupo 1 = 2,4 ± 3,2; Grupo 2 = 5,5 ± 3,4). Não foi observada diferença entre os grupos na concentração de citocinas. CONCLUSÕES: A injeção de 25 mg de S(+)-cetamina por via peridural antes da incisão reduziu a intensidade da dor apenas 12 horas após a incisão cirúrgica e não alterou a concentração de citocinas.

BACKGROUND AND OBJECTIVES: Some studies showed that ketamine inhibits the production of cytokines. The objective of this study was to evaluate the preemptive analgesic effect of epidural S(+)-ketamine in hysterectomy and plasmatic cytokines (IL-6, TNF-α and IL-10). METHOD: A double-blinded study with 29 patients was conducted. Patients in Group 1 received 13 mL of 0.25 percent bupivacaine with 25 mg of S(+)ketamine 30 minutes before surgical incision and 15 mL of saline solution via the epidural route 30 minutes after. Patients in Group 2 received 15 mL of saline solution 30 minutes before the surgical incision, followed by 13 mL of 0.25 percent bupivacaine with 25 mg of S(+)-ketamine 30 minutes after. Postoperative analgesia was made with epidural bupivacaine and fentanyl. Dipyrone 1 g was used whenever required. The following paramenters were evaluated: concentration of cytokines, intensity of pain, time of first request of analgesic and total quantity of analgesic used. RESULTS: Time for the first request for analgesics was 61.5 minutes in Group 1 and 69.0 in Group 2, without difference between these groups. There was no difference for total dose of fentanyl used in Group 1 (221.4 µg) and Group 2 (223.3 µg). A similar analgesic effect was obtained in both groups, except in T12 (Group 1 = 2.4 ± 3.2; Group 2 = 5.5 ± 3.4). No differences in concentration of cytokines were observed. CONCLUSIONS: The epidural injection of 25 mg S(+)-ketamine before incision reduced the pain intensity only 12 hours after surgical incision and did not alter concentration of cytokines.

JUSTIFICATIVA Y OBJETIVOS: Algunos estudios han demostrado que la cetamina inhibe la producción de citocinas. El objetivo de este estudio, fue evaluar el efecto analgésico de prevención y citocinas plasmáticas (IL-6, TNF-α y IL-10) de S(+)-cetamina por vía epidural en la histerectomía. MÉTODO: Fue realizado un estudio doble ciego en 29 pacientes. Pacientes del Grupo 1 recibieron 13 mL de bupivacaína al 0,25 por ciento con 25 mg de S(+)-cetamina 30 minutos antes de la incisión quirúrgica, y 15 mL de solución salina fisiológica 30 minutos después de la incisión por vía epidural. Pacientes del Grupo 2, recibieron 15 mL de salina 30 minutos antes de la incisión quirúrgica, seguido de 13 mL de bupivacaína al 0,25 por ciento, más 25 mg de S (+)-cetamina 30 minutos después. La analgesia postoperatoria se realizó con bupivacaína y fentanil por vía epidural. Cuando fue necesario, se usó 1 g de dipirona. Se evaluaron: la concentración de citocinas, la intensidad del dolor, el tiempo de la primera solicitación del analgésico, y la cantidad total de analgésico utilizado. RESULTADOS: El tiempo para la primera solicitación de analgésico fue de 61,5 minutos en el Grupo 1 y 69,0 en el Grupo 2, sin haber diferencias entre los grupos. No hubo diferencias entre los grupos para la dosis total de fentanil usada en el Grupo 1 (221,4 µg) y en el Grupo 2 (223,3 µg). Se obtuvo un efecto analgésico parecido en los grupos, con excepción en T12 (Grupo 1 = 2,4 ± 3,2; Grupo 2 = 5,5 ± 3,4). No fue observada diferencia entre los grupos en la concentración de citocinas. CONCLUSIONES: La inyección de 25 mg de S(+)-cetamina por vía epidural antes de la incisión, redujo la intensidad del dolor solamente 12 horas después de la incisión quirúrgica y no alteró la concentración de citocinas.

Citocinas e dor: [revisão] / Cytokines and pai: [review]

JUSTIFICATIVA E OBJETIVOS: As citocinas são substâncias necessárias para a resposta inflamatória, favorecendo a cicatrização apropriada da ferida. No entanto, a produção exagerada de citocinas pró-inflamatórias a partir da lesão pode manifestar-se sistemicamente com instabilidade hemodinâmica ou distúrbios metabólicos. O objetivo desta revisão foi descrever os efeitos das citocinas na dor. CONTEÚDO: Este artigo faz uma revisão dos efeitos das citocinas na dor. Em doenças que cursam com processo inflamatório agudo ou crônico, as citocinas podem ser reconhecidas por neurônios e utilizadas para desencadear diversas reações celulares que influenciam na atividade, proliferação e sobrevida da célula imunológica, bem como na produção e atividade de outras citocinas. As citocinas podem ser pró-inflamatórias e anti-inflamatórias. As pró-inflamatórias estão relacionadas com a fisiopatologia das síndromes dolorosas. Foram descritas as células que secretam as citocinas, as citocinas pró-inflamatórias (IL-1, IL-2, IL-6, IL-7 e FNT) e anti-inflamatórias (IL-4, IL-10, IL-13 e FTCβ), as funções de cada citocina e como ocorre a ação dessas substâncias no processamento da dor. CONCLUSÕES: As citocinas desempenham importante papel na dor, agindo através de diferentes mecanismos em vários locais das vias de transmissão da dor.

BACKGROUND AND OBJECTIVES: Cytokines are necessary for the inflammatory response, favoring proper wound healing. However, exaggerated proinflammatory cytokine production can manifest systemically as hemodynamic instability or metabolic derangements. The objective of this review was to describe the effects of cytokines in pain. CONTENTS: This article reviews the effects of cytokines in pain. In diseases with acute or chronic inflammation, cytokines can be recognized by neurons and used to trigger several cell reactions that influence the activity, proliferation, and survival of immune cells, as well as the production and activity of other cytokines. Cytokines can be proinflammatory and anti-inflammatory. Proinflammatory cytokines are related with the pathophysiology of pain syndromes. Cells that secrete proinflammatory (IL-1, IL-2, IL-6, IL-7, and TNF) and anti-inflammatory (IL-4, IL-10, IL-13, and TGFβ) cytokines, the functions of each cytokine, and the action of those compounds on pain processing, have been described. CONCLUSIONS: Cytokines have an important role in pain through different mechanisms in several sites of pain transmission pathways.

JUSTIFICATIVA Y OBJETIVOS: Las citocinas son sustancias necesarias para la respuesta inflamatoria, favoreciendo la cicatrización apropiada de la herida. Sin embargo, la producción exagerada de citocinas proinflamatorias a partir de la lesión puede manifestarse sistémicamente con la inestabilidad hemodinámica o disturbios metabólicos. El objetivo de esta revisión fue describir los efectos de las citocinas en el dolor. CONTENIDO: Este artículo intenta hacer una revisión de los efectos de las citocinas en el dolor. En enfermedades que se manifiestan con un proceso inflamatorio agudo o crónico, las citocinas pueden ser reconocidas por las neuronas y utilizadas para desencadenar diversas reacciones celulares que influyen en la actividad, proliferación y sobrevida de la célula inmunológica, como también en la producción y en la actividad de otras citocinas. Las citocinas pueden ser proinflamatorias y antiinflamatorias. Las proinflamatorias tienen una relación con la fisiopatología de los síndromes dolorosos. Ya se han descrito las células que segregan las citocinas, las citocinas proinflamatorias (IL-1, IL-2, IL-6, IL-7 y FNT) y antiinflamatorias (IL-4, IL-10, IL-13 y FTCβ), las funciones de cada citocina y también cómo ocurre la acción de esas sustancias en el proceso del dolor. CONCLUSIONES: Las citocinas desempeñan un rol muy importante en el dolor, actuando por medio de diferentes mecanismos en varios locales de las vías de transmisión del dolor.

Neurotoxicity Screening in a Multipotent Neural Stem Cell Line Established from the Mouse Brain

Neural stem cells (NSCs) have mainly been applied to neurodegeneration in some medically intractable neurologic diseases. In this study, we established a novel NSC line and investigated the cytotoxic responses of NSCs to exogenous neurotoxicants, glutamates and reactive oxygen species (ROS). A multipotent NSC line, B2A1 cells, was established from long-term primary cultures of oligodendrocyte-enriched cells from an adult BALB/c mouse brain. B2A1 cells could be differentiated into neuronal, astrocytic and oligodendroglial lineages. The cells also expressed genotypic mRNA messages for both neural progenitor cells and differentiated neuronoglial cells. B2A1 cells treated with hydrogen peroxide and L-buthionine-(S,R)-sulfoximine underwent 30-40% cell death, while B2A1 cells treated with glutamate and kainate showed 25-35% cell death. Cytopathologic changes consisting of swollen cell bodies, loss of cytoplasmic processes, and nuclear chromatin disintegration, developed after exposure to both ROS and excitotoxic chemicals. These results suggest that B2A1 cells may be useful in the study of NSC biology and may constitute an effective neurotoxicity screening system for ROS and excitotoxic chemicals.

Induction of Macrophage Migration Inhibitory Factor in ConA-Stimulated Rheumatoid Arthritis Synovial Fibroblasts through the P38 MAP Kinase-Dependent Signaling Pathway

BACKGROUND/AIMS: This study was undertaken to identify the intracellular signaling pathway involved in induction of macrophage migration inhibitory factor (MIF) in human rheumatoid arthritis (RA) synovial fibroblasts. METHODS: Human RA synovial fibroblasts were treated with concanavalin A (ConA), various cytokines, and inhibitors of signal transduction molecules. The production of MIF by synovial fibroblasts was measured in culture supernatants by ELISA. The expression of MIF mRNA was determined using reverse transcriptase polymerase chain reaction (RT-PCR) and real-time PCR. Phosphorylation of p38 mitogen-activated protein (MAP) kinase in synovial fibroblasts was confirmed using Western blotting. The expression of MIF and p38 MAP kinase in RA synovium was determined using dual immunohistochemistry. RESULTS: The production of MIF by RA synovial fibroblasts increased in a dose-dependent manner after ConA stimulation. MIF was also induced by interferon-gamma, CD40 ligand, interleukin-15, interleukin-1beta, tumor necrosis factor-alpha, and transforming growth factor-beta. The production of MIF by RA synovial fibroblasts was significantly reduced after inhibition of p38 MAP kinase. The expression of MIF and p38 MAP kinase was upregulated in the RA synovium compared with the osteoarthritis synovium. CONCLUSIONS: These results suggest that MIF production was induced through a p38 MAP-kinase-dependent pathway in RA synovial fibroblasts.

Mediators and mechanisms of pancreatic beta-cell death in type 1 diabetes

Type 1 diabetes mellitus (T1D) is characterized by severe insulin deficiency resulting from chronic and progressive destruction of pancreatic beta-cells by the immune system. The triggering of autoimmunity against the beta-cells is probably caused by environmental agent(s) acting in the context of a predisposing genetic background. Once activated, the immune cells invade the islets and mediate their deleterious effects on beta-cells via mechanisms such as Fas/FasL, perforin/granzyme, reactive oxygen and nitrogen species and pro-inflammatory cytokines. Binding of cytokines to their receptors on the beta-cells activates MAP-kinases and the transcription factors STAT-1 and NFkappa-B, provoking functional impairment, endoplasmic reticulum stress and ultimately apoptosis. This review discusses the potential mediators and mechanisms leading to beta-cell destruction in T1D.

O diabetes melito tipo 1 (DM1) tem como característica uma grave deficiência de insulina que resulta da destruição da célula-beta, crônica e progressiva, pelo sistema imune. O desencadeamento da autoimunidade contra a célula-beta é causado, provavelmente, por agentes ambientais que atuam quando existe predisposição genética. Uma vez ativadas, células imunes invadem as ilhotas, e os efeitos deletérios sobre as células-beta são mediados por mecanismos relacionados a Fas/FasL, perforina/granzima, espécies reativas de oxigênio e nitrogênio, e a citocinas pró-inflamatórias. A ligação de citocinas a seus receptores na célula-beta ativa MAP-quinase e fatores de transcrição STAT-1 e NFkapaB, provocando prejuízo funcional, estresse de retículo endoplasmático e, por fim, apoptose. Esta revisão discute os mecanismos e os mediadores potenciais que levam à destruição da célula-beta no DM1.

Uso de corticosteróides em cirurgia / Use of corticosteroids in surgery

Introdução: o efeito antinflamatório dosglicocorticóides tem implicações clínicas que interferem de forma decisiva na evolução de pacientes submetidosa procedimentos cirúrgicos. Objetivos: realizar uma revisão sobre o uso adequado de corticosteróides emcirurgia. Métodos: Foram coletados e analisados artigos referentes ao emprego de corticoterapia em pacientescríticos, bem como pacientes submetidos a procedimentos eletivos. Todos os artigos foram extraídos do banco de dados Medline.Discussão: Apesar de seusinúmeros efeitos colaterais, é clara na literatura a participação dos corticosteróides na modulação doprocesso inflamatório sistêmico durante uma situação de estresse. A administração de tais drogas durante oprocedimento cirúrgico deriva do seu emprego prévio em pacientes em sepse e cujo prognóstico sofreuconsiderado impacto após sua indicação em maior escala. Conclusões: Concluímos que o uso de corticosteróides nas doses e intervalos definidos promove benefícios a homeostasia dos pacientes submetidos aprocedimento cirúrgicos, bem como, evolução pósoperatória com menor morbidade .

Background: the anti-inflamatory effect of the glicocorticoids have clinical implication in the evolution of patients that go under surgical procedures. Objective: Review the appropriate use of glucocorticoids in surgery.Methods: articles focused in corticotherapy in critically illpatients as well as corticotherapy in patients that go under elective procedures were selected and analyzed. All the articles were selected from Medline data base. Discussion:Despite of its large number of collateral effect, it’s well defined in the literature the use of corticosteroids in themodulation of the systemic inflammatory process along a situation of stress. The administration of this drugs alongthe surgical procedure comes from its usage in patients with sepsis which prognosis suffered important impact after its indication in large scale. Conclusions: we concluded that the use of corticosteroids in doses and defined period of time has benefits in the homeo stasis of patients submitted to surgical procedures as well as a less morbidityin the postoperative time .

The effects of fluconazole and cytokines on human mononuclear cells

Candida infections are common infections and fluconazole is one of the most frequently administered antifungal agents in their treatment. The resistance developed against antifungal agents has necessitated the improvement of new treatments. This study focuses on the investigation of the effect of fluconazole and cytokines such as interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF) on chemokine production and anticandidal activity of human monocytes. In the study it was observed that GM-CSF caused an increase in candidacidal activity of monocytes. Anticandidal activity of GM-CSF + IFN-gamma combination was not found to be more effective than GM-CSF or IFN-gamma alone. The presence of cytokine and fluconazole caused an increase in the levels of CCL3 and CCL4 chemokines. Accordingly, it was considered that chemokines could contribute to the efficacy of fluconazole in C. albicans infections. Besides, in order to strengthen the immune system some cytokines might be used in addition to antifungal agents for the treatment.

Differential Expression, Shedding, Cytokine Regulation and Function of TNFR1 and TNFR2 in Human Fetal Astrocytes

Tumor necrosis factor (TNF) -alpha induces pleiotropic cellular effects through a 55kDa, type 1 receptor (TNFR1) and a 75kDa type 2 receptor (TNFR2). Moreover, it participates in the pathogenesis of several CNS diseases, including demyelinating diseases. TNF- receptors are differentially expressed and are regulated in many cell types. However, data regarding the TNF-alpha receptor expression and regulation in human astrocytes is limited to date. We investigated TNF-alpha receptor expression, its regulation by cytokines, and its functional role in primary cultured human fetal astrocytes, which are the most abundant cellular population in the central nervous system and are known to be immunologically active. In this study, astrocytes were found to constitutively and predominantly transcribe, translate and shed TNFR1 rather than TNFR2, but TNFR2 expression was increased by adding TNF-alpha, IL-1, and IFN-gamma, but not by adding LPS. To determine the functional roles of TNFR1 and TNFR2 on TNF induction, we investigated NF-kappaB activation and TNF-alpha induction after neutralizing TNFR1 and TNFR2 by an antibody treatment. We found that NF-kappaB activation and TNF-alpha induction are blocked by TNFR1 neutralizing antibody treatments.

Isolation of Endothelial Progenitor Cells from Cord Blood and Induction of Differentiation by Ex Vivo Expansion

Endothelial progenitor cells (EPCs) have been reported to possess the capacity to colonize vascular grafts and hold promise for therapeutic neovascularization. However, limited quantities of EPCs have been the major factor impeding effective research on vasculoangiogenesis. In this study, cytokine and culture conditions necessary for the provision of large quantities of endothelial cells (ECs) were investigated. Cord blood was collected from 18 normal full-term deliveries and CD34+ cells were isolated by MACS system (Miltenyi Biotech, Bergish-Gladbach, Germany). To evaluate the effect of cytokines, CD34+ cells were cultured with various cytokine combinations, such as stem cell factor (SCF), flt3-ligand (FL), and thrombopoietin (TPO) with vascular endothelial growth factor (VEGF), interleukin-1beta, fibroblast growth factor-basic (FGF-b) as basic cytokines. The quantities of non-adherent and adherent cells were the greatest with SCF, FL and TPO. The addition of TPO to all other cytokines significantly increased the number of non-adherent and adherent cells (p< 0.05, Wilcoxon rank sum test). After four weeks of culture, adherent cells expressed endothelial specific markers such as KDR, CD31 and CD62E. Typical morphology of ECs was observed during culture, such as cord-like structure and cobblestone appearance, suggesting that the adherent cells were consistent with ECs. In this study, the experimental conditions that optimize the production of ECs for therapeutic neovascularization were described. And it was possibly suggested that TPO plays a major role in differentiation from EPCs to ECs.

Isolation of ethanol-induced genes in pancreatic beta-cells by representational difference analysis (RDA)

Recent epidemiological studies suggest that alcohol consumption is one of the risk factors leading to type 2 diabetes, but the direct effect of ethanol on beta-cell gene expression is not known. Here, using cDNA RDA method, we isolated 43 ethanol-induced genes in pancreatic beta-cells, and confirmed their differential expression by Northern blot or semi-quantitative RT-PCR. These genes were further categorized by the functional criteria based on the published data; Translation, Transcription, Metabolism, Signal transduction, Transport, Structure, Cytoskeleton, Regulation, or Putative/Unknown genes. The effects of each gene on beta-cell function need to be further investigated, however, the present data strongly suggest that these genes might be related to the metabolic alterations caused by ethanol as indicated in earlier study. In particular, RPS3 gene expression was increased by ethanol, glucosamine, and cytokines, implying that ethanol might decrease the metabolic activity by oxidative stress in beta-cells. Therefore, cloning of these genes in full-length and the detailed studies of each gene on beta-cell functions might provide clues on the pathophysiology of type 2 diabetes caused by alcohol.

Expression and regulation of integrin receptors in human trophoblast cells: role of estradiol and cytokines.

Embryo implantation and placentation are dynamic cellular events that require not only synchrony between the maternal environment and the embryo, but also complex cell-cell communication amongst the implanting blastocyst and the receptive endometrium through integrins, a large family of proteins involved in the attachment, migration, invasion and control of cellular functions. Integrins display dynamic temporal and spatial patterns of expression by the trophoblast cells during early pregnancy in humans. However, the precise mechanism of embryo implantation and the modulation of the integrin receptors during blastocyst attachment and further implantation remain elusive in the humans. The present study elucidates the expression and hormonal modulation of fibronectin, vitronectin and laminin integrin receptors by estradiol and IL-1alpha in human trophoblast cells. Human first trimester trophoblast cells showed the induction of the classical estrogen receptor (ER)-alpha by its own ligand, estradiol. Treatment with either estradiol or IL-1alpha induced the expressions of alpha4, alpha5, alpha6 and alpha(v) integrin receptor subunits at both the mRNA and protein levels, while expression of beta1 remained unaltered. Furthermore, estradiol upregulated the expression of IL-1alpha, thereby suggesting the possibility that estrogen may either directly or via the proinflammatory cytokine induces the expression of the cell surface integrin receptors. The findings delineate the role of hormones and the cytokines in modulating the adhesiveness and attachment of the trophoblast cells. This may reflect the in vivo scenario where the implanting embryo is surrounded by a hormone-cytokine rich uterine microenvironment that may precisely regulate the expression of integrins and thereby facilitate implantation.

B cells activated in the presence of Th1 cytokines inhibit osteoclastogenesis

Host immune response has been considered as an important disease-modifying factor of periodontitis, however, which immune cell(s) or factor(s) are involved in the destruction of periodontium remains unclear. Previously, we reported that osteoclastogenesis is enhanced by activated B cells but suppressed by activated CD8(+)T cells. We present new data that B cells activated in the presence of Th1 cytokines inhibit osteoclastogenesis. Purified murine B cells were activated with anti-IgD mAb, IL-4, and anti-CD40 mAb, in the absence (B(Th2)) or presence of Th1 cytokines, either IL-2 (B(IL-2)) or IFN-gamma (B(IFN-gamma)). Each activated B cell population was co-cultured with RAW264.7 cells in the presence of soluble receptor activator of NF-kappaB ligand (sRANKL), and the effect on osteoclastic differentiation was evaluated. While B(Th2)increased osteoclastogenesis, B(IL-2)and B(IFN-gamma)suppressed it profoundly. To verify the mediating molecule(s), we analyzed cytokine profiles of the activated B cells. Compared to B(Th2), B(IL-2)expressed increased amount of IFN-gamma and B(IFN-gamma)expressed decreased amounts of IL-4, IL-5, and IL-10. IFN-gamma was a key negative regulator of osteoclastic differentiation, and mediated the inhibition by B(IL-2). These results suggest that Th1 cytokines may have new important roles in resistance to periodontitis, acting directly on osteoclasts or indirectly through B cells.

Anti-angiogenic activity of BIM (bio-immunomodulator).

Angiogenesis and immune suppression are the two important factors responsible for embryo implantation and development of tumor. Therefore, disruption of angiogenesis and upliftment of immune function is essential for control of tumor growth as well as to regulate the activity of post coital contraceptives. BIM is an immunomodulatory cytokine obtained from rodent bone marrow that showed anti-implantation and anti-tumor activities. It also improved T/B cell and monocyte macrophage functions. In this communication the anti-angiogenic property of BIM is demonstrated in pregnant rat model. BIM induced disintegration of uterine myometrium and blood vessels. No implantation was observed compared to control. It is proposed that depending upon the physiological condition of the host BIM could modulate host immune function and exert its anti-angiogenic effect.