Dissertations / Theses on the topic 'Tumor necrosis factor Physiological effect'

Includes bibliographical references (leaves 182-240). Explores alternate strategies that may alter inflammatory cytokine production, particularly tumour necrosis factor đ [tumor necrosis factor-alpha], and therefore provide a possible treatment for rheumatoid arthritis.

Thesis (M.A.)--University of North Carolina at Chapel Hill, 2008.
Title from electronic title page (viewed Dec. 11, 2008). "... in partial fulfillment of the requirements for the degree of Master of Arts in the Department of Exercise and Sport Science Exercise Physiology." Discipline: Exercise and Sports Science; Department/School: Exercise and Sport Science. On t.p. and in abstract, [alpha] is Greek letter.

TNF defers apoptosis in macrophages undergoing spontaneous or pharmacologically (thapsigargin, ceramide, CCCP, etoposide or cisplatin)-induced apoptosis, as determined by measurements of caspase-3 activity and annexin-V staining (Chapter 2). The action requires TNF interaction with TNF-R1, not TNF-R2. Survival is uniquely reliant on the activity of the NF-B signaling pathway, and does not require activities arising from the PI3K/Akt, JNK, ERK, p38 MAP kinase or iNOS pathways (Chapter 3). Further, the general anti-apoptotic property of TNF and its specific antagonism of CCCP-induced apoptosis led to the finding that TNF action prevents cytochrome c release. This protection is likely mediated through effects on components of the MPTP itself, as TNF exhibited functional redundancy with the pore inhibitor cyclosporin A, and did not modify upstream events that promote MPTP opening during apoptosis, namely ROS production, cytosolic Ca2+ increase, or a reduction of total ATP (Chapter 4). Subsequent experiments with the mRNA synthesis inhibitor, actinomycin D, and the translation inhibitor, cycloheximide revealed that the protein(s) responsible for TNF-induced survival was transcribed and translated within 1 hr. However, western analyses provided no convincing evidence of the involvement of Mn-SOD, cIAP-1, XIAP, Bcl-2 or A1 in TNF cytoprotection (Chapter 5). Rather, microarray experiments identified the consistent induction of an early response gene, pim-1, within 30 min of TNF exposure (Chapter 6). This result was verified at the protein level with a specific Pim-1 antibody. Evidence was also found for induction of the anti-apoptotic protein A20, but only at mRNA level. Parthenolide, wortmannin, SP600125, PD98059, SB203580 or L-NAME1 acted against TNF-induced Pim-1 expression in a pattern that exactly matched the effects of these inhibitors on TNF-induced survival. That is, only parthenolide-mediated inactivation of NF-B abolished TNF-induced induction of Pim-1. TNF also stimulated the rapid phosphorylation (inactivation) of the pro-apoptotic BH3-only protein, Bad at Ser112 in a manner sensitive to NF-B inhibition, but not PI3K/Akt, JNK, ERK or p38 MAP kinase inhibition (Chapter 7). As Bad is a known substrate of Pim-1 and Bad 1 Parthenolide, wortmannin, SP600125, PD98059 and SB203580 are inhibitors of the NF-B, PI3K/Akt, JNK, ERK and p38 MAP kinase pathways, respectively. L-NAME inhibits iNOS. NF-B- and mitochondria-linked signaling events that contribute to TNF action in deferring physiological and chemotherapeutic drug-induced apoptosis in macrophages ii phosphorylation occurred coincident with Pim-1 upregulation, it is likely that Pim-1 kinase activity mediates the inactivation of Bad. The overall data therefore supports a model in which TNF ligation of TNF-R1 at the cell surface results in intracellular NF- B activation, leading to the induction of Pim-1 mRNA and protein, and the ensuing phosphorylation of Bad. Inactivation of pro-apoptotic Bad increases the resistance threshold of mitochondria to apoptotic insults, thereby reducing the occurrence of mitochondrial permeability transition, cytochrome c release and subsequent caspase-3 activation.

The present study examines the role of liver macrophages (Kupffer cells), of C57BL/6 mice, as effector cells responsible for the killing of Entamoeba histolytica trophozoites in vitro. Interferon gamma (IFN-$ gamma$) and tumor necrosis factor alpha (TNF) were each shown to endow murine Kupffer cells with significant amoebicidal activity. Interferon gamma alone was not able to activate Kupffer cells to amoebicidal state. However, IFN-$ gamma$ and lipopolysaccharide (LPS) acted synergistically in this phenomenon. It seems that the acquisition of amoebicidal activity is associated with the involvement of hydrogen peroxide, because the addition of catalase partially decreases the killing of this parasite by Kupffer cells. In addition, it appears that the amoebicidal activity of IFN-$ gamma$-treated Kupffer cells is contact-dependent.
In our study, Kupffer cells were also shown to be activated by TNF in vitro to an amoebicidal state and this cytolytic effect depends upon the ratio of Kupffer cells to amoebae, the concentrations of TNF used, and the time of exposure of the cells and the parasites to TNF.
Our results indicate that the immunologic production of IFN-$ gamma$ and TNF is important in the activation of Kupffer cells for controlling this parasite and that Kupffer cells are strong effector cells against the amoebae.

Adipose tissue (AT) expansion and remodeling that maintains healthy function relies on stromal preadipocytes capable of differentiating into new adipocytes (adipogenesis). During chronic positive energy balance, a relative deficit in adipogenesis, from either a decrease in preadipocyte number or their capacity to differentiate, leads to excessive adipocyte hypertrophy and AT dysfunction. AT contains macrophages whose number and activation state is dynamically regulated with changes in AT mass. This study aims to investigate the effect of macrophage-secreted factors on preadipocyte survival. To assess the effect of macrophage-secreted factors on preadipocytes, murine 3T3-L1 preadipocytes or human primary preadipocytes were incubated with macrophage-conditioned medium (MacCM), prepared from either murine (J774A.1, RAW264.7, bone marrow-derived) or human (THP-1, monocyte-derived) macrophage models, respectively. MacCM inhibited preadipocyte apoptosis and activated pro-survival signaling in both preadipocyte models. Inhibition of PDGFR, Akt, or ERK1/2 reduced the pro-survival effect of MacCM in 3T3-L1 preadipocytes. Inhibition of reactive oxygen species (ROS) generation, or enhancement of ROS clearance, reduced MacCM-dependent 3T3-L1 preadipocyte survival. Whereas anti-inflammatory activated macrophages retained the ability to prevent preadipocyte apoptosis, pro-inflammatory activated macrophages did not. TNF-α immunoneutralization restored the survival activity of pro-inflammatory MacCM on 3T3-L1 preadipocytes. These studies reveal a novel pro-survival effect of MacCM on preadipocytes, and identify signaling molecules (PDGF, Akt, ERK1/2, and ROS) that underlie this action. Macrophage activation was found to regulate the pro-survival activity of MacCM. These in vitro cell culture studies are consistent with a model in which the extent of preadipocyte apoptosis in vivo may determine preadipocyte number and the ability of AT to expand while maintaining healthy function during chronic positive energy balance.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
A cirurgia para remoÃÃo de terceiros molares constitui-se um procedimento frequentemente realizado em odontologia, estando associado a variados graus de dor pÃs-operatÃria, podendo afetar a qualidade de vida dos pacientes. Considerando o benefÃcio mÃximo ao paciente submetido a uma cirurgia, insere-se a analgesia preemptiva como estratÃgia farmacolÃgica amplamente pesquisada nas Ãltimas dÃcadas. O objetivo do presente estudo foi avaliar o efeito da analgesia preemptiva sob os efeitos inflamatÃrios e sob os nÃveis de citocinas prÃ-inflamatÃrias (TNF-α e IL-1β) em cirurgias de terceiros molares inferiores. Foi realizado um estudo unicÃntrico, triplo-cego, randomizado, placebo-controlado, com 36 pacientes submetidos à remoÃÃo cirÃrgica de terceiros molares mandibulares (n=72) que foram randomicamente alocados para receber etoricoxibe 120 mg, ibuprofeno 400mg ou placebo 1 hora prÃ-operatoriamente, e os eventos inflamatÃrios (dor, edema e abertura bucal) foram avaliados. Houve diferenÃa significativa entre os grupos com relaÃÃo aos escores de dor (p<0,001). Etoricoxibe e ibuprofeno reduziram os escores de dor em relaÃÃo ao placebo (p<0,05). A dosagem de TNF-α do grupo placebo nÃo mostrou diferenÃa estatisticamente significante (p=0,127) do tempo 0â para o tempo 30â minutos, enquanto que o ibuprofeno e o etoricoxibe mostraram reduÃÃo significativa entre os tempos. A dosagem de IL-1β dos grupos placebo e etoricoxibe nÃo mostraram variaÃÃo significativa, porÃm, no grupo ibuprofeno houve reduÃÃo significante (p=0,038) dos nÃveis do tempo 0` para o tempo 30`. Como conclusÃo do estudo, os nÃveis de TNF-α e IL-1β, bem como os eventos inflamatÃrios em cirurgias para remoÃÃo de terceiros molares inferiores, mostraram-se inversamente proporcionais à seletividade COX-2 do AINE utilizado preemptivamente, e estes apresentaram reduÃÃo significativa dos parÃmetros clÃnicos referentes aos eventos inflamatÃrios em comparaÃÃo ao grupo placebo.

Orientador: Edson Rosa Pimentel
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-26T15:25:06Z (GMT). No. of bitstreams: 1 Vieira_CristianoPedrozo_D.pdf: 3245048 bytes, checksum: 0556c35183a4503cef8427390d086781 (MD5) Previous issue date: 2015
Resumo: Nutrição terapêutica é a administração de alguns nutrientes, em doses maiores que as necessidades alimentares diárias que podem prevenir deficiências orgânicas e atuar como agentes farmacológicos. A glicina apresenta amplos efeitos benéficos em processos inflamatórios e tumorais. O Chá verde feito de folhas e brotos da planta Camellia sinensis, é a segunda bebida mais consumida em todo mundo. O interesse econômico e social tem ganhado espaço no mercado e atualmente seu consumo faz parte da rotina diária de muitas pessoas que utilizam essa bebida como uma finalidade terapêutica. O Chá verde possui propriedades antimutagênicas, antidiabéticos, antiinflamatórias, antioxidante, antimicrobial e hipocolesterolêmica. A tendinite é reconhecidamente um problema clínico que motiva a comunidade científica a buscar tratamentos que auxiliem no restabelecimento das propriedades funcionais dos tendões. O presente estudo investigou o efeito do chá verde e ou da ração rica em glicina após 7 e 21 dias da indução da tendinite com colagenase. Ensaios bioquímicos, moleculares, morfológicos e biomecânicos foram desenvolvidos. Além disso, tenócitos em cultura foram tratados com glicina após inflamação induzida por TNF-?. Nossos ensaios in vivo mostraram altas concentrações de hidroxiprolina e glicosaminoglicanos no grupo glicina e chá em 21 dias de tratamento. Nos ensaios biomecânicos os grupos chá verde e dieta de glicina em 21 dias suportaram maiores cargas biomecânicas antes da ruptura. Além disso, uma melhor organização das fibras de colágeno foi observada no grupo chá verde em 7 dias. Análises bioquímicas e moleculares da junção miotendínosa mostraram que a inflamação instalada na região osteotendinea pode provocar alterações significativas nesse local. Marcantes alterações foram notadas nas metaloproteínases (MMP) tais como MMP-2, MMP-8 e MMP-9 em animais com tendinite tratados ou não com chá verde e glicina. No estudo in vitro, tenócitos extraídos a partir de tendão de Aquiles foram tratados com TNF-?, seguindo ou não de tratamento com glicina em meio de cultura. Antes e após 24 horas da inflamação foi adicionado glicina. Tenócitos inflamados e tratados com glicina mostraram expressão de colágeno tipo I próxima aos grupos tratados com glicina previamente e depois da inflamação quando comparado ao grupo controle. Todos os grupos tratados com glicina mostraram menor expressão de MMP-2. A atividade da MMP-9 foi alta apenas no grupo tratado com glicina em 48 horas. A concentração de ácido urônico foi menor no grupo tratado com glicina 24 horas após a inflamação. No ensaio de migração celular, resultados em 24 horas de tratamento foram similares ao grupo controle. Em geral, tanto a glicina quanto o chá verde influenciam na síntese dos componentes do tendão, melhoram a organizaçao das fibras colagênicas, aumentam a resistência a cargas do tendão inflamado e consequentemente aceleram o processo de remodelamento após indução da tendinite. Além disso, o tratamento com glicina em cultura de tenócitos mostrou uma reorganização eficiente da matriz extracelular, corroborando com os resultados encontrados in vivo
Abstract: Therapeutic nutrition is the administration of some nutrients, in higher doses than those recommended for the daily food needs that can prevent dysfunctions and act as pharmacological agents. Glycine has large beneficial effects in inflammatory and tumor processes. Green tea made from leaves and buds of the Camellia sinensis plant, is the second most consumed beverage in the world. The economic and social interest has gained space in the market and currently its consumption is part of the daily routine of many people who use this drink as a therapeutic purpose. Green tea has antimutagenic, antidiabetic, anti-inflammatory, antioxidant, antimicrobial and hypocholesterolemic properties. Tendinitis is recognized as a clinical problem that motivates the scientific community to investigate treatments that help in restoring the functional properties of tendons. The present study investigated the effect of green tea and/or diet rich in glycine after 7 and 21 days of tendinitis collagenase-induced. Biochemical, molecular, morphological and biomechanical tests were developed. Furthermore, tenocytes in culture were treated with glycine after inflammation induced by TNF-?. Our tests in vivo showed high concentrations of hydroxyproline and glycosaminoglycans in glycine and green tea group in 21 days of treatment. In biomechanical assay, green tea and glycine diet groups in 21 days showed a high biomechanical loads bore before rupture. In addition, better organization of collagen fibers was observed in green tea group in 7 days. Biochemical and molecular analyzes of myotendinous junction showed that the inflammation installed in osteotendinious region can cause significant change in that region. Remarkable changes were noted in metalloproteinases (MMP) such as MMP-2, MMP-8 and MMP-9 in animals with tendinitis treated with or without glycine and green tea. In the in vitro study, tenocytes from Achilles tendon were treated with TNF-?, or not following treatment with glycine in the culture medium. Before and 24 hours after inflammation was added glycine. Tenocytes inflamed and treated with glycine showed expression of collagen type I close to the treated groups with glycine previously and after the inflammation when compared to the control group. All treated groups showed less glycine MMP-2 expression. The activity of MMP-9 was high only in the group treated with glycine for 48 hours. In the cell migration assay results in 24 hours of treatment were similar to the control group. In general, both glycine and green tea influenced the synthesis of the tendon components, improve the organization of the collagenous fibers, increase the load resistance of the inflamed tendon and consequently accelerate the remodeling process after inducing tendinitis. In addition, the treatment with glycine in tenocytes culture showed efficient reorganization of the extracellular matrix, confirming the results found in vivo
Doutorado
Biologia Celular
Doutor em Biologia Celular e Estrutural

Amendments inserted inside front cover.
Bibliograpghy: leaves 111-139.
xvi, 139, [29] leaves, [10] leaves of plates : ill. ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
The in vivo adminstration of Tumour Necrosis Factor-alpha as an antineoplastic agent has been severely restricted by dose-limiting side effects. Human TNF mutants with selective binding to the Human TNF receptors were employed to examine the role of these receptors in the mediation of TNF's cytotoxic and proinflammatory activities.
Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1995?

Amendments inserted inside front cover.
Bibliograpghy: leaves 111-139.
xvi, 139, [29] leaves, [10] leaves of plates : ill. ; 30 cm.
The in vivo adminstration of Tumour Necrosis Factor-alpha as an antineoplastic agent has been severely restricted by dose-limiting side effects. Human TNF mutants with selective binding to the Human TNF receptors were employed to examine the role of these receptors in the mediation of TNF's cytotoxic and proinflammatory activities.
Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1995?

Explores alternate strategies that may alter inflammatory cytokine production, particularly tumour necrosis factor α [tumor necrosis factor-alpha], and therefore provide a possible treatment for rheumatoid arthritis.
Thesis (Ph.D.) -- University of Adelaide, Dept. of Medicine, 2001

Copies of author's previously published articles inserted.
Includes bibliographical references.
vii, 39 leaves : ill. ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Shows that the cytokine tumour necrosis factor [alpha] (TNF-[alpha]) enhances the adhesion of neutrophils to the endothelium by an action both on the neutrophil and on the endothelial cell.
Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1995?

Copies of author's previously published articles inserted.
Includes bibliographical references.
vii, 39 leaves : ill. ; 30 cm.
Shows that the cytokine tumour necrosis factor [alpha] (TNF-[alpha]) enhances the adhesion of neutrophils to the endothelium by an action both on the neutrophil and on the endothelial cell.
Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1995?

Obesity-induced inflammation has been linked to the onset of insulin resistance (IR), which is a strong risk factor for the development of T2DM. Recent studies have indicated that tumor necrosis factor alpha (TNFα) plays a causative role in obesity-mediated IR via its overexpression in adipose tissue. Moreover, TNFα has been shown to induce the IR and the alteration of lipid and glucose metabolism in adipose tissue at various levels including normal adipocyte differentiation and mature adipocyte function. However, the mechanisms linking TNFα-induced adipocyte dysfunction in chronic obesity to the IR and T2DM are largely unknown. Herein we report that TNFα inhibited the mRNA expression of PPARγ, which is a key nuclear transcriptional factor for driving preadipocyte differentiation and maintaining normal function of mature adipocyte. TNFα treatment suppressed preadipocyte differentiation by downregulating mRNAs for FAS, perilipin, GLUT4, adiponectin, PGC-1α and C/EBPα and also altered adipocyte function by inhibiting mRNAs for perilipin, GLUT4, CPT-1 and PGC-1α, while increasing the expression of IL-6 and MCP1 mRNAs. In palmitic acid (PA)-induced in vitro hypertrophic adipocytes, we found that mRNA expression of inflammatory cytokines (TNFα and IL-6) was significantly elevated, while the expression of mRNAs for PPARγ, perilipin and adiponectin was markedly reduced, suggesting that TNFα may induce dysfunctional adipocyte phenotypes by targeting PPARγ and its target genes. In in vivo study, mice fed high fat diet (HFD) for 16 weeks had adipose tissue dysfunction as reflected by significant reduction of protein expression for PPARγ, perilipin, FAS and FABP4, consistent with the results observed in in vitro hypertrophic adipocytes. Interestingly, this was reversed by the loss of TNFα in TNFα knockout mice, indicating that TNFα may induce adipocyte dysfunction via the inhibition of PPARγ and its target genes. In the liver, HFD significantly increased hepatic triglyceride (TG) contents, while decreasing de novo lipogenesis (SCD1 and FAS), whereas TNFα deficiency decreased TG content and de novo lipogenesis compared to HFD-fed wild-type (WT) mice, suggesting that TNFα-induced adipocyte dysfunction is associated with hepatic lipid deposition in chronic obesity. Taken together, the current findings provide new insights into the role of TNFα in obesity-induced inflammation and also suggest the TNFα as a mediator for obesity-induced IR & T2DM

by Lam Kai Yi.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1998.
Includes bibliographical references (leaves 156-165).
Abstract also in Chinese.
Chapter Chapter One: --- Introduction --- p.1
Chapter 1.1 --- Tumor Necrosis Factor-α in Cancer Treatment --- p.1
Chapter 1.1.1 --- Historical Background --- p.1
Chapter 1.1.2 --- Mechanisms of Action --- p.2
Chapter 1.1.2.1 --- Production of Reactive oxidative Species
Chapter 1.1.2.2 --- Increase of Intracellular Free Calcium Concentration
Chapter 1.1.2.3 --- Activation of Ca2+/Mg2+-dependent Endonuclease
Chapter 1.1.2.4 --- Decrease of glucose uptake and Protein Synthesis
Chapter 1.1.2.5 --- Formation of Ion-permeable Channel
Chapter 1.1.2.6 --- Activation of Phospholipase
Chapter 1.1.2.7 --- Increase of S-phase Cells
Chapter 1.1.2.8 --- Immunomodulatory Effects
Chapter 1.1.3 --- Resistance of Cells to TNF-α --- p.7
Chapter 1.1.4 --- Clinical Studies --- p.11
Chapter 1.1.5 --- Side Effects --- p.12
Chapter 1.2 --- Hyperthermia and Cancer Treatment --- p.14
Chapter 1.2.1 --- Hyperthermic Agents --- p.15
Chapter 1.2.2 --- Intrinsic Heat Sensitivity --- p.15
Chapter 1.2.3 --- Mechanisms of Action --- p.17
Chapter 1.2.3.1 --- Depolarization of Membrane Potential
Chapter 1.2.3.2 --- "Reduction of glucose transport and DNA, mRNA and Protein Synthesis"
Chapter 1.2.3.3 --- Decrease of Intracellular pH
Chapter 1.2.3.4 --- Calcium Imbalance
Chapter 1.2.3.5 --- Effect on Nucleolar Protein
Chapter 1.2.3.6 --- Apoptosis
Chapter 1.2.3.7 --- Induction of Autologous Tumor Killing
Chapter 1.2.3.8 --- "Blood Flow, Tumor Oxygenation and Vascular Damage"
Chapter 1.2.4 --- Clinical Studies --- p.20
Chapter 1.3 --- Combined Treatment --- p.21
Chapter 1.3.1 --- Combined Treatment with TNF-α and Fixed-temperature Hyperthermia --- p.22
Chapter 1.3.2 --- Combined Treatment with TNF + Step-down Hyperthermia --- p.22
Chapter 1.3.3 --- In Vivo Study --- p.23
Chapter 1.3.4 --- Sequence of Treatment --- p.24
Chapter 1.3.5 --- Proposed Mechanism of Synergism --- p.24
Chapter 1.4 --- Objective of Study --- p.26
Chapter 1.4.1 --- Sequence of Treatments --- p.26
Chapter 1.4.2 --- Comparison of Treatments' Effectiveness --- p.27
Chapter 1.4.3 --- Effect on Normal Cell --- p.27
Chapter 1.4.4 --- Effect on Distribution of Cells in Cell Cycle Phases --- p.28
Chapter 1.4.5 --- In Vivo Study --- p.28
Chapter Chapter Two: --- Materials and Methods --- p.30
Chapter 2.1. --- Materials --- p.30
Chapter 2.1.1 --- For Cell Culture --- p.30
Chapter 2.1.2 --- In vitro Treatments --- p.31
Chapter 2.1.3 --- DNA Electrophoresis --- p.31
Chapter 2.1.4 --- Flow Cytometry --- p.32
Chapter 2.2. --- Reagent Preparation --- p.33
Chapter 2.2.1 --- Culture Media --- p.33
Chapter 2.2.2 --- Human Recombinant Tumor Necrosis Factor alpha (rhTNF-α) --- p.33
Chapter 2.2.3 --- Phosphate Buffered Saline (PBS) --- p.33
Chapter 2.2.4 --- Lysis Buffer --- p.34
Chapter 2.2.5 --- TE Buffer --- p.34
Chapter 2.2.6 --- Proteinase K and Ribonuclease A (RNase A) --- p.34
Chapter 2.2.7 --- 100 Base-Pair DNA Marker --- p.34
Chapter 2.2.8 --- Propidium Iodide (PI) --- p.35
Chapter 2.3 --- Methods --- p.35
Chapter 2.3.1 --- Cell Culture --- p.35
Chapter 2.3.1.1 --- Ehrlich Ascitic Tumor (EAT) and Human Leukemia (HL-60)
Chapter 2.3.1.2 --- Human Coronary Artery Endothelial Cells (HCAEC)
Chapter 2.3.2 --- In vitro Experiments --- p.36
Chapter 2.3.3 --- Tumor Necrosis Factor Treatment --- p.37
Chapter 2.3.4 --- Hyperthermia Treatments --- p.37
Chapter 2.3.5 --- Cell Counting --- p.38
Chapter 2.3.5.1 --- Trypan Blue Exclusion Assay
Chapter 2.3.5.2 --- Neutral Red Assay
Chapter 2.3.6 --- Determination of Additive or Synergistic Effect --- p.39
Chapter 2.3.7 --- DNA Electrophoresis --- p.40
Chapter 2.3.8 --- Flow Cytometry --- p.42
Chapter 2.3.7.1 --- Preparation of Samples
Chapter 2.3.7.2 --- Flow Cytometry Acquisition
Chapter 2.3.7.3 --- Analysis
Chapter 2.3.9 --- In vivo Experiments --- p.44
Chapter 2.3.8.1 --- Animal Strain
Chapter 2.3.8.2 --- Cell Line
Chapter 2.3.8.3 --- Tumor Necrosis Factor Treatment
Chapter 2.3.8.4 --- Hyperthermia Treatments
Chapter 2.3.8.5 --- Test of Body Temperature
Chapter 2.3.8.6 --- Cell Harvesting
Chapter Chapter Three: --- Result --- p.50
Chapter 3.1 --- Optimal Sequence of Treatments --- p.50
Chapter 3.1.1 --- Optimal Sequence of Treatments on Murine Ehrlich Ascitic Tumor (EAT) cells --- p.50
Chapter 3.1.1.1 --- TNF + Fixed-temperature Hyperthermia
Chapter 3.1.1.2 --- TNF + Step-down Hyperthermia2
Chapter 3.1.1.3 --- TNF + Step-down Hyperthermia3
Chapter 3.1.2 --- Optimal Sequence of Treatments on Human Leukemia cells HL-60 --- p.60
Chapter 3.1.2.1 --- TNF + Fixed-temperature Hyperthermia
Chapter 3.1.2.2 --- TNF + Step-Down Hyperthermia2
Chapter 3.1.2.3 --- TNF + Step-Down Hyperthermia3
Chapter 3.2 --- Comparison of Effectiveness of Treatments --- p.72
Chapter 3.2.1 --- Effectiveness of Various treatments on EAT cells --- p.72
Chapter 3.2.2 --- Synergistic Effect between rhTNF-α and Hyperthermia on EAT cells --- p.74
Chapter 3.2.3 --- Decrease of Relative Growth and Viability of EAT with Time --- p.79
Chapter 3.2.3.1 --- TNF + Fixed-temperature Hyperthermia
Chapter 3.2.3.2 --- TNF + Step-down Hyperthermia2
Chapter 3.2.3.3 --- TNF + Step-down Hyperthermia3
Chapter 3.2.4 --- Comparison of Effectiveness of Various Treatments on HL-60 cells --- p.82
Chapter 3.2.5 --- Synergistic Effect between rhTNF-α and Hyperthermia on HL-60 cells --- p.87
Chapter 3.2.6 --- Change of Relative Growth and Viability of HL-60 with Time --- p.90
Chapter 3.2.6.1 --- TNF + Fixed-temperature Hyperthermia
Chapter 3.2.6.2 --- TNF + Step-down Hyperthermia2
Chapter 3.2.6.3 --- TNF + Step-down hyperthermia3
Chapter 3.3 --- Cell Death Pathway --- p.96
Chapter 3.3.1 --- Experiments on Ehrlich Ascitic Tumor (EAT) Cells --- p.96
Chapter 3.3.2 --- Experiments on Human Leukemia (HL-60) Cells --- p.100
Chapter 3.4 --- Experiment on Normal Cell --- p.104
Chapter 3.5 --- Effect of TNF + Fixed-temperature Hyperthermia on the Cell Cycle Progression --- p.107
Chapter 3.5.1 --- Different Times of TNF Administration and Distribution of EAT cells in Cell cycle --- p.107
Chapter 3.5.2 --- Different Times of TNF Administration and Distribution of HL-60 cells in Cell Cycle --- p.114
Chapter 3.5.3 --- Shift of Cells Cycle after TNF Treatment --- p.120
Chapter 3.5.3.1 --- Response of Ehrlich Ascitic Tumor Cells
Chapter 3.5.3.2 --- Response of Human leukemia Cells
Chapter 3.6 --- Effectiveness of Treatments in vivo: --- p.129
Chapter 3.6.1 --- Dose-dependent Response --- p.129
Chapter 3.6.2 --- Change of Body Temperature During Hyperthermia --- p.131
Chapter 3.6.3 --- Comparison of Effectiveness of Various Treatments in vivo --- p.133
Chapter 3.6.4 --- Synergistic Effect Between rhTNF-α and Hyperthermia in vivo --- p.135
Chapter Chapter Four: --- Discussion --- p.138
Chapter 4.1 --- Optimal Sequence of Treatments --- p.139
Chapter 4.2 --- Comparison of Various Treatments --- p.143
Chapter 4.3 --- Distribution of Cells in Cell Cycle Phases --- p.149
Chapter 4.4 --- In vivo Study --- p.153
Chapter Chapter Five: --- References --- p.156

碩士
中國醫藥大學
營養學系碩士班
98
Several lines of evidence indicate that inflammation and endothelial cell dysfunction are important initiating events in atherosclerosis. Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, induces the expression of cell adhesion molecules and results in monocyte adherence and atheromatous plaques formation. Andrographolide (AP) is a major bioactive diterpene lactone in Andrographis paniculata, which possesses anti-inflammatory property. In the present study, we investigated the effect of AP on TNF-α-induced ICAM-1 expression in EA.hy926 cells. AP inhibited TNF-α-induced ICAM-1 mRNA, total protein, and cell-surface expressions. In addition, TNF-α-induced adhesion of HL-60 to EA.hy926 cells was abolished by AP. Furthermore, AP suppressed TNF-α-induced κB inhibitor (IκB) kinase (IKK) and IκBα activation, p65 nuclear translocation, NF-κB and DNA binding activity, and promoter activity of ICAM-1. AP increased intracellular cAMP concentration and induced the phosphorylation of cAMP response element-binding protein (CREB). Transfection with CREB-specific small interfering RNA knocked down CREB expression, however, did not inhibit ICAM-1 expression by AP. Taken together, these results suggest that AP down-regulates TNF-α-induced ICAM-1 expression via attenuation of activation of NF-κB in EA.hy926 cells. The results may implicate the cardiovascular-protective potential of AP.

Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2019
A espondilite anquilosante é a mais comum entre as doenças espondiloartriticas e tem uma prevalência entre 1,0-1,4% na população mundial, e 1,6% entre a população de Portugal. A patogénese da EA inclui sinovite, entesite com mecanismos controversos do desenvolvimento da doença que são caracterizados por uma formação óssea local excessiva, também associada à perda sistémica óssea. A inflamação articular resulta em inchaço que causa dor, perda de mobilidade, rigidez articular e danos ósseos. Atualmente, são desenvolvidas estratégias terapêuticas mais eficazes, principalmente para evitar danos ósseos permanentes, causados por esta doença, para que um número maior de pacientes possa entrar em remissão. O fator de necrose tumoral (TNF) é uma citocina que é responsável pela mediação de processos inflamatórios e atividades de imunorregulação, acabando por possuir um papel muito central neste tipo de patologias. Na patologia de EA, o TNF para além de ser um mediador inflamatório sistémico é também responsável pela modulação da renovação óssea. Estudos passados revelaram que níveis séricos dessa citocina foram encontrados mais elevados em pacientes diagnosticados com EA. Os tecidos sinoviais e estesiais dos pacientes com EA também têm sido identificados com elevados valores de TNF. Desta forma o TNF vai desempenhando na EA um papel pró-osteoclastogénico e anti-osteoblastogénico, propondo a existência de uma resposta local através da regulação positiva dos osteoclastos (OCs) e osteoblastos (OBs) (células responsáveis pela reabsorção, formação e mineralização óssea). A presença de células do sistema imunológico e a expressão de citocinas pró-inflamatórias na entese de pacientes com EA já foram relatadas em estudos passados, no entanto, os mecanismos que levam a este acontecimento são pouco compreendidos. Potencializando, a necessidade de desenvolver novos estudos para compreender mais aprofundadamente a EA. Existem também vários estudos que consideram o stress mecânico como um fator estimulante no desencadear deste tipo de doenças. Alguns estudos em ratinhos sugeriram que o stress mecânico poderia estar associado a esta patologia. Estes estudos observaram que comportamentos agressivos e condições de alojamento demonstraram afetar a expressão da doença. Assim como através da imobilização mecânica das patas anteriores e suspensão da cauda, demonstrou menor progressão da doença. No entanto existe falta de provas concretas em que o stress mecânico afeta diretamente o desenvolvimento desta doença. A terapia de inibição do TNF (TNFi) tem sido extensivamente investigada e as terapias têm demonstrado ser frequentemente utilizadas no tratamento de algumas doenças reumatológicas, não atuando apenas na redução da inflamação, mas também na interrupção da destruição articular. Dados de ensaios clínicos, onde pacientes com EA ativa reportam não apenas redução da dor inflamatória nas costas, mas adicionalmente melhoria da mobilidade física, fadiga e qualidade de vida. Foi também demonstrado que a terapia com TNFi é capaz de bloquear parcialmente os mecanismos envolvidos nos danos ósseos. No entanto ainda não foi comprovado que este tratamento consegue interromper a nova formação de osso em EA. Existe ainda a necessidade de estudos adicionais para poder responder ou elucidar os diferentes pontos acima mencionados. Consequentemente ratinhos são frequentemente utilizados como modelos de estudos para a biologia humana, devido a existência de semelhanças genéticas e fisiológicas entre as espécies. Por este motivo a EA é estudada principalmente em modelos animais, uma vez que existe dificuldades na obtenção de amostras sinoviais e de enteses de pacientes. Assim como as amostras periféricas das articulações da coluna vertebral são particularmente as mais difíceis de obter. Neste estudo foi então utilizado um modelo animal que representa a patologia de espondiloartrite (SpA) através da expressão da forma transmembranar de TNF tmTNF(TgA86). Este estudo tem como primeiro objetivo abordar a caracterização deste modelo animal no desenvolvimento desta patologia, segundo avaliar o impacto do stress mecânico neste modelo animal e terceiro analisar o efeito causado pela administração da terapia de TNFi. No decorrer deste estudo foram realizadas avaliações clínicas, seguidas de análise de imagens histológicas marcadas com Safranina e Hematoxilina/Eosina através de uma aplicação de scores semiquantitativos. Ao caracterizar o desenvolvimento desta doença no modelo animal a avaliação inicial clínica mostrou que os ratinhos TgA86 desenvolveram a doença espontaneamente entre as 3 e 4 semanas de idade, com 100% de penetrância e sem perda de peso, conforme descrito na literatura. Este modelo de ratinho mostrou também exibir as características de SpA de poliartrite inflamatória, edema articular e deformações nas patas posteriores e anteriores. A cauda quando foi medida e comparada com a dos ratinhos saudáveis, mostrou-se mais curta e com deformidades, apresentando características da doença. A análise histológica confirmou estes resultados clínicos, através da análise estatística que apresentou desenvolvimento da doença nas patas anteriores, coluna e cauda, onde, entre as articulações axiais, a cauda desenvolveu a patologia primeiro e com mais severidade quando comparada com a coluna. Estes resultados foram provavelmente devido à fisiologia do animal onde a distribuição do seu peso ocorre sobretudo sobre as patas traseiras, onde a cauda desenvolve mecanismos biomecânicos de equilíbrio corporal. Globalmente os resultados mostraram que a severidade da doença aumentou gradualmente com o tempo, através do agravamento da inflamação, erosão óssea e da degradação da cartilagem. Particularmente os discos intervertebrais são severamente danificados na cauda, entesites e proeminente infiltrado inflamatório são também observados. Relativamente ao stress mecânico os ratinhos foram submetidos a um protocolo de exercicio físico utilizando a metodologia acima descrito. Os nossos resultados mostraram que, em geral, o stress mecânico, afeta o desenvolvimento da doença nos ratinhos que expressam a doença. Nestes, a doença expressa-se numa forma mais severa em comparação com os ratinhos saudáveis, bem como os ratinhos que expressam a doença e que não foram submetidos ao exercício. Estes dados estão em linha com os nossos resultados da avaliação clínica realizados durante o protocolo de treadmill. Especialmente nas articulações periféricas, onde os ratinhos exercitados e que expressam a doença, apresentam um aumento da inflamação e danos na cartilagem. Estes resultados surgem através da observação da presença de eritema, inchaço, deformações e perda de funcionamento articular. Em resumo, estes dados fornecem um vínculo entre a biomecânica e a inflamação onde esta é promovida em locais específicos do sistema músculo-esquelético na existência de EA. Consequentemente, o stresse mecânico é sentido pelas células pró-inflamatórias nas regiões articulares, o que indica que nesses locais, as forças biomecânicas são então traduzidas para sinais bioquímicos, como quimiocinas, sendo estas responsáveis pela desencadear de sinais inflamatórios locais podendo conduzir a destruição óssea. No que diz respeito à análise do efeito da terapia inibidora de TNF um equivalente para ratinho de certolizumab pegol (CZP) foi administrado. O modelo de ratinho TgA86 foi submetido a um tratamento precoce às 4 semanas de idade e um tratamento tardio às 10 semanas de idade. As análises histológicas das articulações periféricas (patas traseiras) mostraram uma diminuição significativa da atividade da doença quando ambos os tratamentos foram administrados. No entanto, os resultados demonstram através de comparações entre os dois períodos de tratamento realizados, que o mais precoce mostrou ser mais eficiente no controlo e prevenção do desenvolvimento da doença. Isto pode ser explicado pelo fato de os ratinhos TgA86 com 10 semanas de idade já apresentarem a doença estabelecida. O que se torna problemático na restauração da estrutura óssea, pois os danos existentes podem ser já irreversíveis, devido ao comportamento agressivo da doença. A administração da terapia mostrou a nível axial (cauda e coluna) e periférico (pata) conseguir controlar efetivamente a inflamação sinovial, bem como prevenir os ossos e articulações da degradação, neste modelo animal. Embora o mecanismo de ação completo do TNFi não esteja completamente compreendido, este trabalho trouxe informações sobre os eventos locais que ocorrem nas articulações periféricas e axiais após a administração do tratamento. Em sumário os nossos resultados demonstram que o modelo animal TgA86 apresenta manifestações semelhantes a EA e apresenta desenvolvimento e progressão da doença ao longo do tempo, sugerindo que este modelo animal em geral tende a ser um bom modelo para estudos futuros. Adicionalmente concluímos que o stresse mecânico pode ser considerado um fator contribuinte para o agravamento desta doença neste modelo animal. Relativamente à terapia de inibição do TNF foi observado que a sua eficácia é dependente do estado de desenvolvimento da doença no momento da administração. Os nossos resultados demonstram que a eficácia terapêutica do inibidor de TNF é superior quando administrado numa fase inicial da doença.
Ankylosing spondylitis is the most common among spondyloarthritic diseases and has a prevalence between 1.0-1.4% in the world population, and 1.6% within Portugal population. The pathogenesis of AS includes synovitis, enthesitis with controversial mechanisms of disease development that are characterized by excessive local bone formation, and at the same time it is also associated with systemic bone loss. The joint inflammation results in swelling which causes pain, loss of mobility, joint stiffness and damage. Currently, more effective therapeutic strategies are being developed, mainly to avoid permanent bone damage caused by this disease, so that a greater number of patients can enter into remission. The tumor necrosis factor (TNF) is a cytokine responsible for the mediation of inflammatory processes and immunoregulatory activities, being proposed to play a very central role in this type of pathology. In AS pathology, TNF, besides being a systemic inflammatory mediator, is also responsible for the modulation of bone remodelling. Past studies have shown that serum levels of this cytokine were found to be higher in patients diagnosed with AS. Synovial and enthesial tissues of AS patients also have been identified with high levels of TNF. Therefore TNF plays a pro-osteoclastogenic and anti-osteoblastogenic role in AS, proposing the existence of a local response through the positive regulation of osteoclasts (OCs) and osteoblasts (OBs) (cells responsible for bone resorption, formation and mineralization). The presence of immune system cells and expression of pro-inflammatory cytokines at the enthesis of AS patients was previously shown, however the mechanisms leading to it are poorly understood. Contributing for the development of new studies to improve the AS pathology acknowledgement. There are also several studies that consider stress as trigger in this type of disease. Some studies in mice have suggested that mechanical stress could be associated with this pathology. These studies observed that aggressive behaviours and housing conditions affected the expression of the disease. Others through mechanical strain of the hind paws and suspension of the tail showed less disease progression. However, there is a lack of concrete evidence that mechanical stress can directly affect the development of this disease. The therapy of TNF inhibition (TNFi) has been extensively investigated and shown to be frequently used in the treatment of some rheumatologic diseases, acting not only in reducing inflammation but also in stopping joint destruction. Data from clinical trials, where patients with active AS reported not only reduction of inflammatory back pain, but also improvement of mobility, fatigue and quality of life. It has also been shown that TNFi therapy is able to partially block the mechanisms involved in bone damage. However, it has not yet been proven that this treatment can interrupt new bone formation in AS. Translating to the necessity of further studies taking place to be able to answer or elucidate the subjects mentioned above. Consequently mice are often used as study models for human biology because of the existence of genetic and physiological similarities between species. For this reason, AS is mainly studied in animal models, since there are difficulties in obtaining patients’ synovial and enthesis samples of peripheral and particularly of the spine joints. In this study an animal model was selected and used, that represents the pathology of spondyloarthritis (SpA) through the expression of the transmembrane form of TNF tmTNF(TgA86). The objective of this study is first to approach the characterization of this disease development in this animal model, second to evaluate the impact of mechanical stress on this animal model and third to analyse the effect caused by the administration of TNFi therapy. During this research work, clinical evaluations were performed, followed by analysis of histological images stained with Safranin, Haematoxylin and Eosin (H&E) through the application of semi-quantitative scores. When characterizing the development of this disease in this animal model, the initial clinical evaluation showed that the TgA86 mice spontaneously developed the disease between 3 and 4 weeks of age, with 100% penetrance and without weight loss, as described in the literature. This mouse model also showed the SpA characteristics of inflammatory polyarthritis, joint erythema and deformation on the paws. When measured and compared with healthy mice, the tail was deformed and shorter, presenting characteristics of the disease. Histological analysis corroborated the clinical results, through statistical analysis that showed development of the disease in the hind paws, spine and tail. Where, between the axial joints, the tail developed earlier and more severally the pathology when compared with the spine. These results were probably due to the own physiology of the animal where the distribution of its weight occurs mainly on the hind paws, and the tail develops biomechanical mechanisms of body balance. Nevertheless, the overall results showed that the severity of the disease increased gradually over time, by increasing inflammation, bone erosion and cartilage damage. Particularly the intervertebral discs are severely damaged in the tail, enthesis and prominent inflammatory infiltrate are also observed. Regarding mechanical stress, the mice were submitted to a treadmill protocol and to the same type of methodology above described. Our results showed that, in general, mechanical stress affects the development of the disease in mice exercised expressing the disease. Where the disease is developed in a more severe form compared to healthy mice, and to mice unexercised that express the disease. Histological analysis in the specific locations of the peripheral joints where mice exercised expressing the disease, showed increased inflammation and cartilage damage. This was observed clinically through the presence of erythema, swelling, deformations and loss of function. In summary, these data provided a link between biomechanics and inflammation where it is promoted in specific sites of the musculoskeletal system in AS pathology. Consequently, mechanical stress is sensed by pro-inflammatory cells in regions of the joints, which indicates that at these sites, biomechanical forces are then translated into biochemical signals, such as chemokines, which promote local inflammation and bone destruction. Regarding to the analysis of the effect of TNF inhibitory therapy, an equivalent for mice of certolizumab pegol (CZP) was administered. The mice model TgA86, was submitted to an early treatment at 4 weeks old and a late treatment at 10 weeks old. Histological analyses of the peripheral joints (hind paws) showed a significant decrease of disease activity when both treatments were administered. However, results also showed through comparisons made between the two treatment phases of the, that the earliest phase had a higher success rate in controlling and preventing the development of disease. This can be explained by the fact that disease is already established in the TgA86 mice at 10 weeks of age. This can be considered problematic when comes to remodelling the bone structure, because the existing damage could be already irreversible, due to the disease aggressive behaviour. The administration of the therapy displayed at both peripheral (paw) and axial level (tail and spine) the ability to effectively control the synovial inflammation, as well as to protect the bone damage in the joints in this animal model. Although the full mechanism of CZP action is not fully understood, this work has brought information on local events occurring in peripheral and axial joints after treatment administration. In summary our results show that TgA86 show AS-like manifestations and progression over time, suggesting that this animal model in general could be a good model for future studies. Additionally, we concluded that mechanical stress can be considered a contributing factor for worsening of this disease in this animal model. On the subject of TNF inhibition therapy, it was observed that its efficacy is dependent on the phase of administration in the disease development. Here our results demonstrate superior efficacy when the drug is administered at an earlier stage.

Thesis (Ph.D.)--State University of New York at Buffalo, 2005.
Title from PDF title page (viewed on May 12, 2006) Available through UMI ProQuest Digital Dissertations. Thesis adviser: Spengler, Robert N., Ignatowski, Tracey A. Includes bibliographical references.

碩士
中山醫學大學
營養學研究所
97
Atherosclerosis is an inflammatory disease in which the early stages of atherosclerosis are initiated by accumulation of oxLDL and activation of endothelial cells with subsequent expression of adhesion molecules and increased recruitment of leukocytes to the vascular endothelium. It is reported that stimulation of macrophages with oxLDL leads to enhanced TNF-α secretion. TNF-α is an inflammatory cytokine, and this implicates that oxLDL is an inflammatory stimulus. Andrographolide is a diterpene lactone isolated from the leaves of the Andrographis paniculata, which has been shown to possess anticancer, hepato-protective, and antiinflammatory activities. This study examined the effects of andrographolide on TNF-α-induced monocyte/human endothelial cell interaction. Andrographolide inhibited the adhesion of HL-60 cells to TNF-α-induced human umbilical vein endothelial cells (HUVECs) and suppressed total protein, cell surface expression and the promoter activity of the intracellular adhesion molecules-1 (ICAM-1). Andrographolide also abolished TNF-α-induced phosphorylation of Akt (p-Akt) in a time-dependent manner, and the ICAM-1 promoter activity decreased when cells pretreated with PI3K inhibitor LY294002. Moreover, andrographolide blocked nuclear translocation and DNA binding activity of NF-κB. In summary, these results demonstrated that andrographolide exhibits an anti-inflammatory property by down-regulating the pathway that affects the expression of ICAM-1 and the action of andrographolide may be closely related to the inhibition of PI3K/Akt and NF-κB activation in HUVEC. This indicates that andrographolide may play an important role in the prevention of inflammatory responses such as the atherosclerosis process.

碩士
靜宜大學
食品營養研究所
98
Hyperglycemia which major characterizes diabetes, may be related to inflammation and oxidative stress. Inflammation and oxidative stress were of the major risk factor for diabetic complications. The purpose of this study was to investigate the effect of high glucose or inflammation on the cell injury and oxidative stress of liver cell. Human hepatocellular carcinoma cell line (HepG2) was cultured with (5, 10, 15, 25, 35 mM) glucose and/or 0, 0.1, 1, 10, 20 ng/ml tumor necrosis factor-α (TNF-α) in the experiment. Cell viability, nitrite content, MDA content, for 24, 72, 120 hr after treatment and NF-κB protein expression were measured. Additional L-ascorbic acid or α-tocopherol (0, 1, 10, 50, 100 μM) was added to measure cell viability and nitrite content. Results were HepG2 cell viability of groups treated with 25, 35 mM glucose and/or 20 ng/ml TNF-α exhibited significant lower than control group (5 mM glucose) in the same culture time. HepG2 cell viability of groups treated with 0.1, 1, 20 ng/ml TNF-α exhibited significant lower than 0 ng/ml TNF-α treated group for 24 hr. HepG2 cell viability of groups treated with 25 mM glucose and 20 ng/ml TNF-α exhibited significant higher than 25 mM glucose treated group for 120 hr. HepG2 cell NF-κB protein expression of groups treated with 35 mM glucose and/or 20ng/ml TNF-α exhibited significant higher than control group (5 mM glucose) for 24 hr, but nitrite content and MDA were not significantly different with each other, except that HepG2 cell nitrite content of groups treated with 10 ng/ml TNF-α exhibited significant lower than control group (0 ng/ml TNF-α) for 120 hr. HepG2 cell viability of groups treated with 100 μM L-ascorbic acid exhibited significant lower than control group (5 mM glucose) for 120 hr; HepG2 cell viability of groups treated with 1, 10 μM α-tocopherol exhibited significant lower than control group (5 mM glucose) for 24 hr; HepG2 cell nitrite product of groups treated with 50, 100 μM α-tocopherol exhibited significant higher than control group (5 mM glucose) for 72 hr. Generally, high glucose concentration induced cell injury, with mechanism was correlated to NF-κB protein expression.

碩士
國立陽明大學
解剖暨細胞生物學研究所
93
Recent large clinical trials have demonstrated that statin（HMG-CoA reductase inhibitor）, markedly reduces morbidity and mortality when used in the primary and secondary prevention of cardiovascular diseases not only by its lipid-lowering potential but also by its nonlipid-related mechanism. It has been reported that statins can counteract TNF-alpha induced downregulation of thrombomodulin（TM）, which plays a critical role in both anticoagulation and anti-inflammation. In this study, Human Aortic Endothelial Cells（HAECs） pretreated with these two different statins, pravastatin and simvastatin, were used to exmine the expression of adhesion molecules, CD40, TM under TNF-alpha induction and their underlying mechanism. We found that pretreatment with these two statins in TNF-alpha -treated HAECs（1） decreased the expression of CD40 but did not affect the expression of ICAM-1 and VCAM-1.（2）increased the expression of TM.（3）Pretreatment of these two statins increased the expression of TM.（4）Pretreatment of these two statins in TNF-alpha -treated HAECs decreased the activation of MAPK, whereas pretreatment of MAPK inhibitirs in TNF-alpha –treated HAECs did not affect the expression of TM. Futhermore, pretreatment with GGTI-286 and Tcd-B in TNF-alpha –treated HAECs increased the expression of TM, in contrast pretreatment with Y-27632 and C3 did not affect the expression of TM, in addition, pretreatment with these two statins in TNF-alpha –treated HAECs decreased the activation of NF-κB. From the alone results, the effects of statins on TM expression may be mediated by the inactivation of Rac1 and Cdc42 than by the inactivation of NF-κB but not though MAPKs pathway. The increase in endothelial cell TM activity in response to statin constitutes a novel pleiotropic（non-lipid-related） effect of these commonly used compounds.

Adipose tissue is a key regulator of energy metabolism and glucose homeostasis by promoting triglyceride storage and breakdown in various physiological states. Obesity, however, alters adipose tissue metabolism, inducing chronic inflammation, followed by excessive lipolysis. This results in higher systemic free fatty acid (FFA) levels, leading to desensitization of insulin signaling and ultimately to insulin resistance. Although the link between obesity and progression of insulin resistance and type 2 diabetes mellitus (T2DM) remains unclear, tumor necrosis factor-alpha (TNF-alpha) has been proposed to be a key player in promoting obesity-related development of T2DM through chronic inflammation of adipose tissue. TNF-alpha has direct and indirect mechanisms by which it elicits insulin resistance in adipocytes. TNF-alpha attenuates insulin signaling by directly inhibiting insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1). Indirectly, TNF-alpha activates signaling pathways to increase lipolysis and FFA release into circulation, leading to insulin resistance. Lipid droplet-associated fat specific protein 27 (FSP27) protects adipocytes from lipolysis by regulating the lipolytic capacity as well as transcription of adipose triglyceride lipase (ATGL). It has been observed that TNF-alpha promotes lipolysis by reducing the expression of FSP27 in murine adipocytes. The effect of TNF-alpha on lipolysis human adipocytes has also been studied; yet its effect on promoting insulin resistance in human adipocytes still remains elusive. In the present study, we examined the effect of FSP27 on TNF-alpha induced lipolysis and insulin resistance in human adipocytes. TNF-alpha enhanced lipolysis in cultured human adipocytes. In addition, TNF-alpha reduced the expression of endogenous FSP7 and the phosphorylation of AKT, inhibiting the activation of insulin signaling pathway in cultured human adipocytes. FSP27 overexpression, however, attenuated TNF-alpha induced lipolysis and restored activation of insulin signaling through phosphorylation of AKT in cultured human adipocytes. Taken together, these data suggest that FSP27 has a protective effect against TNF-alpha induced lipolysis and insulin resistance through regulating lipolysis and insulin signaling in human adipocytes.

This study investigated the association between flavonoid profiles of different cowpea (Vigna Unguiculata) varieties with anti-inflammatory properties as a possible benefit against inflammatory bowel disease. Cowpea, a drought tolerant annual herbaceous legume that originated in Africa, is known to possess high levels of polyphenolics, which have demonstrated anti-inflammatory, immunoregulatory and antioxidant properties. Black, red, white, brown and light brown cowpeas were investigated for phenolic content and composition using UV-Visible Spectroscopy and HPLC; antioxidant activation mechanism (AOX) by oxygen radical absorbance capacity (ORAC). Anti-inflammatory activity was measured via NF-κB activation in Raw 264.7 macrophages challenged with a lipo-polysaccharide. Phenols, tannins and AOX activity were generally similar within phenotypes; however among light brown varieties, 09FCV-CC-27M, had among the highest phenols, tannins and AOX, whereas IAR-48 had among the lowest. White cowpea (EARLY ACRE) variety showed the least amount of total phenol content (78.2 mg GAE/g) and condensed tannin content (4.1 mg CE/g); whereas the red varieties (IT82D-889, IT97K-1042-3) contained the highest amounts of tannins (242 and 132 mg CE/g), and phenols (431 and 454 mg GAE/g) respectively. Antioxidant activity correlated with phenol content data. Anthocyanins were only found in the black cowpea. The red varieties had the highest levels of flavonols, which were mostly quercetin derivatives; the white and light brown (IAR-48) varieties had quercetin-3-O-diglucoside as the dominantcompound. The light brown variety (09FCV-CC-27M) had the highest amount of flavan-3-ols while in the white variety no flavan-3-ols were detected. Unexpectedly, the cowpea extracts with lower phenolic and tannins content, the white and light brown (IAR-48) varieties, showed significant (p<0.05) anti-inflammatory properties in the LPS induced macrophages, inhibiting the activation of NF-κB at different concentrations (0.33, 1.67 and 3.33 μg extract/mL). Conversely, extracts with higher phenolic and tannin content did not induce anti-inflammatory response at similar concentrations, suggesting that tannins or other phenolics interfered with anti-inflammatory response at these concentrations. These results suggest that cowpea composition is an important determinant of anti-inflammatory response in inflammatory bowel disease.

Thesis (Ph. D.)--University of Alabama at Birmingham, School of Joint Health Sciences, 1997.
eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

碩士
中國醫藥學院
中國醫學研究所
86
Bu-zhong-yi-qi-tang (BZYQT) is the kind of popular traditional Chinese medicine, and has been used for the treatment of hepatocellular carcinoma (HCC). At present, we still do not know the effects of BZYQT on the immune system very well. The present in vitro study demonstrated that BZYQT is capable of increasing tumor necrosis factor-a (TNF-a) and granulocyte colony-stimulating-factor (G-CSF) produced by peripheral blood mononuclear cells (PBMC). The production levels of G-CSF by PBMC of health volunteers were higher than those of patients with HCC when more than 625 mg/ml of BZYQT was added. The reason may be due to the impaired immunologic reactivity in patients with HCC. When adding high concentration 3125 mg/ml of BZYQT to the cultured PBMC, the increment of cytokines production decreased although there were no obvious changes in the number of metabolic active PBMC. Further studies are required to clarify this finding. By the way, BZYQT could not influence interferon-g or transforming growth factor b1 produced by PBMC. TNF-a and G-CSF are known to play important roles in the biological defense mechanism. Administration of BZYQT may, therefore, be beneficial for patients with HCC because it can induce these cytokines.

碩士
中國醫藥大學
中國醫學研究所
99
According to the theory of Traditional Chinese medicine, cerebrovascular accident mainly results from blood stasis. Carthamus tinctorious L. (CT) is considered that has the action of activate blood to eliminate stasis since long time ago. Several studies have known recently that CT has antioxidant, ant inflammation and inhibiting glutamate-mediated injury action, and also can protect neuronal cell in the brain. Therefore, the purpose of the present study is to investigate effect of CT on cerebral infarct. A total of 72 male Sprague-Dawley (SD) rats were studied. In experiment one (54 SD), an animal model of cerebral infarct was established by occluding bilateral common carotid arteries (CA) and the right middle cerebral artery (MCA) for 90 min, then reperfusion for 24 hrs. Intra- peritoneal (ip) administration of CT 0.2 g/kg, 0.4 g/kg, 0.6 g/kg and MK801 1.0 mg/kg 10 min before , and CT 0.4 g/kg 30 min after occluding the cerebral blood flow, respectively. In addition, CT 0.4 g/kg i.p. was done 30 min after reperfusion of 2 hrs. The cerebral infarct size and grade of neurological deficit were used as an index to evaluate the effect of CT on cerebral infarct. The superoxide anion was measured by lucigenin- Chemiluminescence (CL) counts before and 90 min after occluding the cerebral blood flow, and 2 hrs after reperfusion, respectively. The tumor necrosis factor-alfa (TNF-α) and interleukin-1beta (IL-1β) immunostaining in the core of cerebral infarct area, and the levels of blood sugar were also measured 24 hrs after reperfusion. The results indicated that pretreatment with CT 0.2 g/kg, 0.4 g/kg, 0.6 g/kg, MK801, and post-treatment with CT 0.4 g/kg all decreased the ratio of cerebral infarct area. Pretreatment with CT 0.4 g/kg or 0.6 g/kg also decreased the grade of neurological deficit. Pretreatment with CT 0.4 g/kg can decrease the lucigenin-CL counts at reperfusion of 2hrs, and it also can decreased the counts of both TNF-α and IL-1β immunostaining positive cells in the cerebral infarct area, but the levels of blood sugar and rectal temperature were similar to the control. In conclusion, CT can decrease both the ratio of cerebral infarct area and grade of neurological deficit, suggesting can be used to treat acute stage of cerebral infarct in humans. This effect of CT has relationship to inhibit superoxide anion to reduce the generation of oxygen free radicals, and decreased proinflammatory cytokine TNF-a and IL-1β resulting to inhibit inflammatory response, but no relationship to blood sugar and rectal temperature were noted. Keywords: Carthamus tinctorious L., Cerebral infarct, Neurological deficit, Superoxide anion, TNF-α、IL-1β

碩士
長庚大學
傳統中國醫學研究所
92
In Traditional Chinese Medicine, the etiology of epileptic seizure is considered that it is due to the imbalance between Yi and Yang in the liver system, resulting in liver wind stirring internally. Therefore, the method of calm the liver to stop the wind is used to treat epileptic seizure. Gastrodia elata Bl.(GE) is one of Chinese herbs, and it is considered that has the action of calm the liver to stop the wind, and is used to treat epileptic seizure for a long period. Our previous studies have known that GE can reduce kainic acid (KA)-induced epileptic seizures in rats, and this effect of GE has closely relationship the suppression or scavenging action of oxygen free radicals. In addition, we also find that GE can inhibit the activation of microglia, therefore, the purpose of the present study to investigate the relationship between anticonvulsive effects of GE and interleukin-1 (IL-1β), tumor necrosis factor-α(TNF-α), and nitric oxide (NO). Animal model of epileptic seizures was performed by KA (12 mg/kg) i.p., and pretreatment with GE 1.0 g/kg, 0.5 g/kg and valproic acid (VA) was done for 4 days, respectively, to observe the effect of GE, the behaviors of the rats and electroencephalogram (EEG) and electromyogram (EMG) were recorded throughout the experimental course. The behavioral observation, EEG and EMG recordings were from 15 min before to 90 min after KA injection. The total counts of wet dog shakes were calculated, and blood of 3 cc was taken from heart for the measurement of IL-1β, TNF-αand NO. Finally, the brains of the rat were taken out for the measurement of IL-1βand TNF-αin the frontal cortex and hippocampus region. The results indicated that both pretreatment of GE 1.0 g/kg and VA 250 mg/kg could reduce the counts of wet dog shakes. The increase of IL-1βand NO in the peripheral blood of KA-treated rats, and these increase can be inhibited by pretreatment with GE 1.0 g/kg or VA 250 mg/kg. In addition, the levels of IL-1βand TNF-αincreased in the frontal cortex, and TNF-αin the hippocampus region in the KA-treated rats, whereas these increase also can be inhibited by pretreatment of GE 1.0 g/kg and VA 250 mg/kg. In conclusion, GE inhibit the generation of IL-1β, TNF-αand NO in the KA-induced epileptic seizures rats, possibly GE mediate via inhibition of microglia activation resulting in IL-1βor TNF-αdecrease to the potent microglia to synthesize inducible nitric synthase (iNOS). Thus the generation of NO and free radicals decreased results in the inhibition of epileptic seizure.