Introduction. Inflammation is characterized by complex interactions between pro- and anti- inflammatory cytokines. Recent research has probed the role of the nervous system in inflammation, part of which includes the cholinergic anti-inflammatory pathway that regulates immunologically-mediated inflammation. In this pathway, norepinephrine release from the splenic nerves binds to beta-2-adrenergic receptors on T cells, causing release of acetylcholine (ACh). ACh subsequently suppresses macrophage production and release of pro-inflammatory cytokines. In this project, we aim to develop a method to quantify the ACh release in splenocytes when challenged with different mediators in this pathway. Our method utilizes liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for quantification of ACh and choline (Ch) in cell culture media. Methods. Literature review revealed that the hydrophilic interaction liquid chromatography (HILIC) is the most appropriate separation mechanism for ACh and Ch. The developed LC-MS/MS method utilizes an isocratic separation (14% 10mM ammonium formate, pH 3, and 86% acetonitrile) on an Atlantis HILIC column (2.1 x 100 mm, 3 micron). The MS operates in positive electrospray (+ESI) mode, monitoring ions specific for ACh, Ch, and their corresponding deuterium labeled internal standards. The calibration range for ACh was 0.5 - 5 micrograms/ml (3.4 - 34 mM) and 10 - 50 micrograms/ml (96 - 480 mM) for Ch. Cell culture media contained neostigmine to inhibit cholinesterase. Cell culture media samples are prepared by freeze drying, reconstituting in acetonitrile, and filtering (0.2micron). Potential loss of ACh through degradation during cell culture was evaluated by monitoring d4-labeled ACh with and without the presence of splenocytes for 4 and 24 hours. Results. Correlation coefficients (R2) indicate linearity for ACh and Ch in acetonitrile and culture media in the aforementioned calibration range. This linearity applies to external calibration as well as calibration utilizing the deuterium labeled internal standard. Calibration curve slopes differed between samples prepared in culture media and those prepared in acetonitrile. As such, experimental samples will be matrix matched by preparing calibrants in media. During the six-min separation, ACh elutes at 3.8 min and Ch at 5.1 min. Deuterium-labeled ACh, when incubated for 4 and 24 hours, showed statistically significant loss, compared to control, of ACh after 24 hours in media and media + splenocytes. Despite this, the average loss of ACh by hydrolysis averaged less than 10%. Media and media + splenocyte samples incubated for 4 hr showed no statistically significant difference from control. Conclusions. The developed LC-MS/MS assay for quantification of ACh and Ch in cell culture media can be applied to the investigation of the cholinergic anti-inflammatory pathway. The method provides a rapid separation of ACh and Ch, with the successful incorporation of stable-isotope labeled internal standards.

Made available in DSpace on 2018-06-27T18:56:33Z (GMT). No. of bitstreams: 2 Gabriela de Moraes Barbosa.pdf: 1970444 bytes, checksum: cc094467f0ae630c8ca1d2412d92b154 (MD5) Gabriela de Moraes Barbosa.pdf.jpg: 3366 bytes, checksum: 682fcf8d3a97c6bde3382a41872a5ae8 (MD5) Previous issue date: 2010-11-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The carbon nanotubes (CNTs) display exclusive properties which are of great interest to biotechnology area. The idea of utilizing CTNs as drug carriers, adjuvant for vaccines and biosensors is attracting much interest from scientific community. However, biocompatibility and toxicity in vitro and in vivo of these structures need to be investigated. The aim of this study was to determine the effects of functionalized multiwalled carbon nanotubes (MWCNTs) on cells of the immune system. For in vitro test, splenocytes and bone marrow cells were isolated from BALB/c female mice. The group of treated cells was cultured with carboxylic MWCNTs suspensions at different concentrations (1, 5 or 10 ng/mL), while the control group was only cultured with medium RMPI 1640 and 10% fetal calf serum (FCS). After 24, 48 and 72 hours of cultivation, the viability proliferation and interaction were analyzed by transmission electron microscopy (TEM) analysis of carboxylic MWCNTs with cells. In vivo study, carboxylic MWCNTs were administered intravenously with a concentration of 50 and 100 &#956;g. After 24 hours, splenocytes were isolated, labeled with anti-CD3+ and anti- B220+ antibodies and analyzed by Fluorescence Activated Cell Sorting (FACS). Total IgG production was determined in mice serum after 20 days of administration. The results showed no significant difference (p < 0.05) in the cell viability test of splenocytes, although it has been observed an increase in the frequency of cells directly proportional to the concentrations of carboxylic MWCNTs suspensions in the period of 48 hours. For marrow cells it was observed an inversion in the frequency of cells in the periods of 24 and 48 hours. Regarding the proliferation of splenocytes, groups of cells treated with carboxylic MWCNTs showed significant difference (p<0.05) when compared to control positive group (CpG oligonucleotide). In the analyses performed by TEM, it was not found any type of interaction of carboxylic MWCNTs with splenocytes treated with carboxylic MWCNTs in the concentration of 10 ng/mL. According to the analyses carried out by flow cytometry, the percentage of CD3+ cells and B220+ cells showed no significant difference in relation to the control group. In Enzyme Immunoassay (ELISA), the total IgG antibody production did not increase in the serum of mice treated with carboxylic MWCNTs when compared to control. Thus, the results in vitro and in vivo indicate that carboxylic MWCNTs did not present stimulatory effects on cells of the immune system and, at the same conditions, no cellular toxicity. / Os nanotubos de carbono (NTCs) apresentam propriedades exclusivas que são de grande interesse para a área de biotecnologia. A idéia de se utilizar NTCs como carreadores de fármacos, adjuvantes para vacinas e biosensores, vem despertando muito interesse da comunidade científica. No entanto, a biocompatibilidade e toxicidade in vitro and in vivo dessas estruturas precisam ser investigadas. O objetivo desse estudo foi determinar os efeitos dos nanotubos de carbono de paredes múltiplas (NTCPMs) carboxilados sobre células do sistema imunológico. Para os testes in vitro, esplenócitos e células da medula óssea foram isolados de camundongos BALB/c fêmeas. O grupo de células tratadas foi cultivado com suspensões de NTCPMs carboxilados em diferentes concentrações (1, 5 ou 10 ng/mL), enquanto o grupo controle foi cultivado apenas com meio RMPI 1640 e 10% soro fetal bovino (SFB). Após 24, 48 e 72 horas de cultivo, foram analisadas viabilidade, proliferação e interação por análise de microscopia eletrônica de transmissão (MET) dos NTCPMs carboxilados com as células. No estudo in vivo, NTCPMs carboxilados foram administrados pela via intravenosa com concentração de 50 e 100 &#956;g. Após 24 horas, os esplenócitos foram isolados, marcados com anticorpos anti-CD3+ e anti-B220+ e analisados por citometria de fluxo. A produção de IgG total foi determinada no soro de camundongos após 20 dias da administração. Os resultados encontrados indicaram que não houve diferença significativa (p<0,05) no teste de viabilidade celular de esplenócitos, embora tenha sido observado um aumento na freqüência de células diretamente proporcional às concentrações das suspensões de NTCPMs carboxilados no tempo de 48 horas. Para as células da medula, foi observada uma inversão na frequência de células nos tempos de 24 e 48 horas. Em relação à proliferação de esplenócitos, os grupos de células tratadas com NTCPMs carboxilados apresentaram diferença significativa (p<0,05) quando comparado ao grupo controle positivo (oligonucleotídeo CpG). Nas análises feitas por MET, não foi encontrado qualquer tipo de interação dos NTCPMs carboxilados com esplenócitos tratados com NTCPMs carboxilados na concentração de 10 ng/mL. De acordo com as análises realizadas por citometria de fluxo, a porcentagem de células de CD3+ e células B220+ não apresentaram diferença significativa com relação ao grupo controle. No teste de Enzima Imunoensaio (ELISA), a produção de anticorpos IgG totais não aumentou no soro de camundongos tratados com NTCPMs carboxilados quando comparada a do grupo controle. Sendo assim, os resultados in vitro e in vivo indicam que NTCPMs carboxilados não apresentam efeitos estimulatórios sobre células do sistema imunológico e, nas mesmas condições, não apresentam toxicidade celular.

Purpose: Inflammation is characterized by complex interactions between pro- and anti- inflammatory cytokines. Recent research has probed the role of the nervous system in inflammation, part of which includes the cholinergic anti-inflammatory pathway that regulates immunologically-mediated inflammation. In this pathway, norepinephrine release from the splenic nerves binds to beta-2-adrenergic receptors on T cells, causing release of acetylcholine (ACh). ACh subsequently suppresses macrophage production and release of pro-inflammatory cytokines. The purpose of this project is to quantify ACh release from isolated murine splenocytes when challenged with different mediators that stimulate T cells in this pathway. Methods:Our method utilizes liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for quantification of ACh and choline (Ch) in cell culture media. The developed LC-MS/MS method utilizes an isocratic separation (14% 10mM ammonium formate, pH 3, and 86% acetonitrile) on an Atlantis HILIC column (2.1 x 100 mm, 3 micron). The MS operates in positive electrospray (+ESI) mode, monitoring ions specific for ACh, Ch, and their corresponding deuterium labeled internal standards. The calibration range for ACh was 0.01 - 5 micrograms/ml (0.068 - 34 mM) and 10 - 50 micrograms/ml (96 - 480 mM) for Ch. Cell culture media contained neostigmine (0.5 mM) to inhibit cholinesterase. Cell culture media samples are prepared by freeze drying, reconstituting in acetonitrile, and filtering (0.2micron). Potential loss of ACh through degradation during cell culture was evaluated by monitoring d4-labeled ACh with and without the presence of splenocytes for 4 and 24 hours. Splenocytes were challenged with saline (control) or 1 mM (-) isoproterenol for 4 and 24 hours in the next set of experiments, and ACh in the medium was quantified. We also evaluated separate and combined effects of isoproterenol and activation of T cells with CD3 and CD28 antibodies on ACh release. Results:Correlation coefficients (R2) indicate linearity for ACh and Ch in culture media in the calibration range. During the six-min separation, ACh elutes at 3.8 min and Ch at 5.1 min. Deuterium-labeled ACh, when incubated in cell culture media for 4 and 24 hours, with and without splenocytes, showed a small but statistically significant loss of ACh after 24 h compared to 0 time media controls. However, the average loss of ACh was less than 10% and was not affected by the presence of splenocytes, suggesting that it was due to chemical hydrolysis. Incubation for 4 hr with and without splenocytes did not affect recovery of ACh. Treatment of splenocytes with isoproterenol for 4 hours did not cause significant release of ACh. However, significant release of ACh was detected after 24 hours exposure to isoproterenol or T cell activation. Media from untreated splenocytes had an ACh concentration of 0.14 +/- 0.07 mcg/mL. Isoproterenol treated had 0.28 +/- 0.14 mcg/mL, T-cell activated had 0.32 +/- 0.17 mg/mL, and isoproterenol + T-cell activation had 0.47 +/- 0.16 mcg/mL. Using a 1-way analysis of variance, statistically significant differences were detected between each of these groups. Conclusion: The developed LC-MS/MS assay for quantification of ACh and Ch in cell culture media can be applied to the investigation of the cholinergic anti-inflammatory pathway in isolated splenocytes. Statistically significant differences in ACh release between control splenocytes and those treated with isoproterenol and T-cell activation can be detected. Quantitative investigation of this pathway helps provide an improved understanding of ACh dynamics as a mediator released from leukocytes. Further studies using this model and methodology will provide novel insights into cholinergic anti-inflammatory mechanisms and other immunomodulatory actions of non-neuronal ACh.

No description available.

The spleen is a key participant in the pathophysiology of sepsis and inflammatory disease. Many splenocytes exhibit a cholinergic phenotype, but our knowledge regarding their cholinergic biology and how they are affected by sepsis is incomplete. We evaluated effects of acute sepsis on the spleen using the cecal ligation and puncture (CLP) model in C57BL/6 and ChATBAC-eGFP mice. Quantification of cholinergic gene expression showed that choline acetyltransferase and vesicular acetylcholine transporter (VAChT) are present and that VAChT is upregulated in sepsis, suggesting increased capacity for release of acetylcholine (ACh). High affinity choline transporter is not expressed but organic acid transporters are, providing additional mechanisms for release. Flow cytometry studies identified subpopulations of cholinergic T and B cells as well as monocytes/macrophages. Neither abundance nor GFP intensity of cholinergic T cells changed in sepsis, suggesting that ACh synthetic capacity was not altered. Spleens have low acetylcholinesterase activity, and the enzyme is localized primarily in red pulp, characteristics expected to favor cholinergic signaling. For cellular studies, ACh was quantified by mass spectroscopy using d4-ACh internal standard. Isolated splenocytes from male mice contain more ACh than females, suggesting the potential for gender-dependent differences in cholinergic immune function. Isolated splenocytes exhibit basal ACh release, which can be increased by isoproterenol (4 and 24 h) or by T cell activation with antibodies to CD3 and CD28 (24 h). Collectively, these data support the concept that sepsis enhances cholinergic function in the spleen and that release of ACh can be triggered by stimuli via different mechanisms.

It is now clear that human and animal exposure to estrogenic compound occurs through several sources. This include: i) naturally occurring endogenous estrogens, ii) exogenous or intentional estrogens for prophylactic (e.g. oral contraceptive) and therapeutic (e.g. as replacement therapy for ovulation in nulliparous women and in menopausal women, and in some men suffering from prostate cancer) purposes, iii) accidental via estrogenic chemical exposure (e.g. pesticides, industrial byproducts) and phytoestrogens (e.g. soybeans). It has long been recognized that estrogen, a female sex hormone, functions not only on the reproductive system, but also on various other systems including the immune system. Estrogens are thought to be of both physiologic and pathologic importance. Female in general, have better immune capabilities than males, a phenomenon attributed to the action of sex hormones on the immune system. There is also a female-gender bias in susceptibility to autoimmune diseases. Estrogens have been linked either directly or indirectly to the etiology and pathogenesis of various female-predominant autoimmune diseases. Estrogens have also been linked to the onset of cancer, and conditions where the immune system often malfunctions. Estrogen affects the functions of both B and T cells, possibly by regulating such factors as lymphokine gene expression and/or cellular death by apoptosis. However, the functioning of both B and T cells under the influence of long-term exposure to estrogen has not been fully understood. The primary aim of this thesis was to investigate the effect of long-term exposure to 17β-estradiol on lymphokine and bcl-2/fas (proto-oncogenes) mRNA expression. We evaluated the effects of estrogen on the expression of genes for lymphokines, which are essential for the immune response. It is hypothesized that estrogen may regulate the immune system by modifying the expression of lymphokine genes and/or genes that regulate apoptosis. The results demonstrated that long-term 17β-estradiol exposure reduced the viability of lymphocytes when compared to lymphocytes from placebo-treated mice. IL-2 and IFN-g mRNA was consistently higher in ConA-stimulated lymphocytes from estrogen-treated mice (P < 0.05). The mRNA for TGF-β₁ lymphokine was also increased but was not consistent at all time points of incubation. The expression of IL-4 mRNA was not noticeably affected by estrogen treatment of mice. Long-term exposure to 17β-estradiol appear to have some influence on the mRNA expression of proto-oncogenes fas and bcl-2 in splenic and thymic T lymphocytes. There was a trend of increased bcl-2 mRNA expression in estrogen-treated mice compared to placebo-treated mice, whereas the mRNA expression of fas gene appeared to be lower compared to controls. Overall, these findings suggest that 17β-estradiol may selectively influence lymphokine and proto-oncogene mRNA expression. These results suggest that the one mode of modulation of the immune response by 17β-estradiol may be through alterations in the lymphokine and proto-oncogene expression. Since estrogen-treatment markedly induces atrophy of the thymus and diminishes the cellularity of the lymphoid organs (e.g. Spleen), it became necessary to perform multiple assays on the same cells, particularly lymphokine and apoptosis gene expression. A secondary objective of this thesis was to investigate whether lymphocytes, which have undergone proliferation in Lympho-Pro™ assay (Alamar Blue assay), could be utilized for further analysis. In this regard, we found that a non-radioactive assay that utilizes Alamar Blue had significant advantages over the conventional ³H-thymidine incorporation assay. By using cells from estrogen and placebo-treated mice in the Alamar Blue assay, we found that this assay not only allowed determination of lymphocyte proliferation, but also the assessment of mRNA expression, cytogenetics, apoptosis and immunophenotyping of the same lymphocytes. / Master of Science

The three insecticides of interest were lindane (an organochlorine), malathion (an organophosphate) and piperonyl butoxide (PBO; a synergist). Based on minimum cytotoxicity (> LC25), the following concentrations were chosen for the pesticide mixture studies: 70μM lindane (Lind), 50μM malathion (Mal) and 55μM PBO. In the AlamarBlue cytotoxicity assay, individual pesticide and mixtures of malathion/PBO (MP) and malathion/lindane (ML) prompted cytotoxicity with varying intensities (Mal 18.8%, Lind 20.4%, PBO 23.5%, ML 53.6% and MP 64.9%). Cytopathological analysis revealed apoptotic features in treated cells and the DNA Ladder Assay confirmed the presence of DNA fragments. The specific mode of cell death was examined via the 7-aminoactinomycin D (7-AAD) Staining Assay. Apoptosis was detected in each treatment (Mal 6.5%, Lind 12.0%, PBO 13.2%, ML 19.3% and MP 23.4%). Furthermore, 7-AAD staining in combination with fluorescent-labeled monoclonal antibodies, PE-CD45RB/220 and FITC-CD90, was performed. B-cells were more susceptible to Mal and PBO treatments than were T-cells. The pro-oxidant activity of the pesticides was monitored via the Dichlorofluorescin Diacetate assay. Exposure to pesticides for 15 minutes increased H2O2 production above the controls, Mal 21.1%; Lind 10.8%; PBO 25.9%; ML 26.8%; MP 37.8%. The activities of antioxidant enzymes, glutathione peroxidase (GSH-Px) and glutathione reductase (GR) were altered by these treatments. GR was significantly reduced for the pesticide mixtures only (control: 51.7; Mal: 48.2; Lind: 50; PBO: 52.3; ML: 40.5; MP: 42 Units/mg). GSH-Px activity was severely reduced for all the pesticide treatments (control: 44.9; Mal: 30.2; Lind: 30.6; PBO: 32.4; ML: 21.1; MP: 21.1 Units/mg). These results indicate that exposure to these pesticide and pesticide mixtures induces apoptosis and oxidative stress. / Master of Science