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Tesis presentada a la Universidad de Chile para optar al grado académico de Magíster en Bioquímica en el área de especialización Toxicología y Diagnóstico Molecular y Memoria para optar al título profesional de Bioquímico / El recambio de elementos celulares en el tejido nervioso es sustentado por la existencia de una célula troncal neural, responsable del proceso de neurogénesis a través del cual se generan neuronas, astrocitos y oligodendrocitos. Recientemente se han identificado algunas quimioquinas con la capacidad de regular el proceso de neurogénesis a partir de una célula troncal neural. En particular, se ha descrito que la alfa-quimioquina CXCL12 y su receptor CXCR4 participan en el desarrollo de estructuras del sistema nervioso central y regulan procesos de proliferación y diferenciación en el cerebro adulto. Sin embargo, el rol que cumplen las alfaquimioquinas y específicamente la quimioquina CXCL12 y sus dos receptores CXCR4 y CXCR7 regulando capacidades funcionales de las células troncales neurales de la médula espinal, aún no está claro. Por lo anterior, esta tesis postula que “las alfa-quimioquinas regulan el proceso de diferenciación de células troncales neurales cultivadas como neuroesferas obtenidas de médula espinal de ratones”. Para evaluar esta hipótesis se utilizaron neuroesferas derivadas de la médula espinal de embriones en estadio E18,5. Se demostró que estas neuroesferas contenían células troncales neurales evaluando las capacidades de autorrenovación y diferenciación. En ellas se determinó que sólo estaba presente el RNA mensajero de la alfa-quimioquina CXCL12 y sus receptores y no el de CXCL1 y sus receptores mediante RT-PCR. De esta manera el estudio se focalizó en CXCL12 y sus receptores CXCR4 y CXCR7. Mediante inmunofluorescencia se detectaron estos receptores en neuroesferas luego de 4 días de diferenciación y se observó que estaban en las células troncales neurales. Finalmente se analizó el proceso de diferenciación, evaluando mediante inmunofluorescencia marcadores de linajes neurales. Se observó que CXCL12 favoreció la diferenciación astroglial sobre la neuronal, comparando la intensidad de fluorescencia de marcadores neurales como GFAP y beta-III-tubulina y que, aparentemente, tanto CXCR4 como CXCR7 participan en el proceso de diferenciación, deducido de los resultados conseguidos con el uso del antagonista de CXCR4, AMD3100. En consecuencia, es posible proponer a la quimioquina CXCL12 como un blanco terapéutico pudiendo bloquearla en casos de lesión a la médula espinal, donde ésta se ve aumentada, para así impedir que las células troncales endógenas se diferencien a astrocitos / The renewal of cells in nervous tissue is sustained by neural stem cells, which are responsible for the neurogenesis that originates neurons, astrocytes and oligodendrocytes. The mechanisms that regulate the functional capabilities of neural stem cells in the spinal cord are unknown. Recently, some chemokines have been identified as regulating factors of neurogenesis of neural stem cells. In particular, the CXCL12 alpha-chemokine and its receptor CXCR4 participate in the development of central nervous system structures and regulate proliferation and differentiation processes in the adult brain. However, the role of alphachemokines and specifically of CXCL12 and its receptors CXCR4 and CXCR7, on the regulation of functional properties of neural stem cells from spinal cord is not clear yet. With this background, this thesis postulates that “alpha-chemokines regulates the differentiation of neural stem cells cultured as neurospheres obtained from mouse spinal cord”. To evaluate this hypothesis, neurospheres from E18,5 mouse spinal cord were used. It was shown that the neurospheres had neural stem cells by assessing their self-renewal and differentiation capabilities. The presence of mRNA of alpha chemokines CXCL1 and CXCL12 and their receptors in neurospheres was evaluated by RT-PCR. Showing that neurospheres have CXCL12 and its receptors mRNA and not CXCL1 and its receptors mRNA. Therefore, the investigation continued with CXCL12 and its receptors CXCR4 and CXCR7. Both receptors were detected on neural stem cells by immunofluorescence after 4 days of differentiation. Finally, the process of differentiation was analyzed by immunofluorescence of neural markers. The comparison between GFAP and beta-III-tubulin fluorescence intensity indicates that CXCL12 promotes astroglial over neuronal differentiation. Apparently, inferred from the use of the CXCR4 antagonist AMD3100, both CXCR4 and CXCR7 participate in the differentiation of the neural stem cells from spinal cord. Given the results obtained in this thesis, it is possible to propose CXCL12 as a potential therapeutic target: blocking it to promote neuronal differentiation, for example, in cases of spinal cord injury, in which CXCL12 mRNA is increased; preventing differentiation of neural stem cells to astrocytes / FONDECYT; MINREB

Glu-Leu-Arg (ELR)-CXC chemokine-mediated neutrophil migration and activation plays a key role in many inflammatory diseases. Dysregulated neutrophil activation often leads to inflammatory responses such as acute lung injury (ALI) or acute respiratory distress syndrome (ARDS).<p> Previously, we generated a bovine drug (i.e., bovine CXCL8(3-74)K11R/G31P, bG31P) by mutating the first two amino acids at the beginning of the N-terminus of bovine CXCL8/IL-8 and later substituting Arg for Lys11 and Pro for Gly31. Bovine G31P was shown to be a highly effective ELR-CXC chemokine and neutrophil antagonist in cattle & guinea pigs, but a human equivalent thereof would be of significantly more use in human medicine. Published studies on the structure and function of human CXCL8 suggest that human CXCL8(3-72)K11R/G31P (i.e., hG31P) would not be a particularly effective chemokine antagonist. Thus, development of a humanized form of bG31P became a primary goal. I first examined the effect of wholesale ligation of the carboxy half of hCXCL8 onto the amino half of bG31P and generated a human-bovine chimeric G31P (hbG31P; i.e., bCXCL8(3-44)K11R/G31P-hCXCL8(45-72)). I also made substitutions at each remaining human-discrepant amino acid (i.e., T3K, H13Y, T15K, E35A, and S37T) within the 5 half of the hbG31P cDNA. The results showed that hbG31P and its analogues blocked CXCL8-induced human neutrophil chemotactic responses, reactive oxygen intermediate (ROI) release, and intracellular calcium flux. Humanized bovine G31P was also shown to significantly block pulmonary neutrophilic pathology in a guinea pig model of airway endotoxemia.<p> As bG31P, hbG31P and its further humanized forms showed essentially equivalent ELR-CXC chemokine antagonist activity, Dr. Fang Li, Ms Jennifer Town and I then generated a fully human form of bG31P, hG31P. <i>In vitro</i>, hG31P was shown to effectively inhibit CXCL-1-, -5-, and -8-induced neutrophil chemotactic responses, intracellular Ca2+ flux, and ROI release. Human G31P also desensitized heterologous G protein-coupled receptors (GPCR) including bacterial peptides (e.g., N-formyl-methionine-leucine-phenylalanine, fMLP), anaphylatoxin (e.g., complement 5a, C5a), lipid mediators (e.g., leukotriene B4, LTB4; platelet-activating factor, PAF) receptors. Moreover, hG31P, in a dose-dependent manner suppressed CXCL1 and CXCL8 expression by LPS-challenged airway epithelial cells and reversed the anti-apoptotic influence of ELR-CXC chemokines on neutrophils. <i>In vivo</i>, hG31P was significantly effective in blocking the pathology associated with airway endotoxemia, aspiration pneumonia, and intestinal ischemia and reperfusion injury, including neutrophil recruitment (70-95% reduction) into, and activation within, the airways or gut, chemokine or cytokine expression, and pulmonary vascular complications. The blockade of neutrophil recruitment by hG31P in aspiration pneumonia animals did not increase airway bacterial growth. The G31P treatment was protective in both mesenteric (i.e., local) and remote organ injury. These findings suggest that hG31P is not only a potent neutrophil antagonist, but an effective blocker of other inflammatory responses. These comprehensive anti-inflammatory effects indicate that hG31P could potentially provide a viable therapeutic approach for inflammatory diseases such as ALI /ARDS.

Glu-Leu-Arg (ELR)-CXC chemokine-mediated neutrophil migration and activation plays a key role in many inflammatory diseases. Dysregulated neutrophil activation often leads to inflammatory responses such as acute lung injury (ALI) or acute respiratory distress syndrome (ARDS).<p> Previously, we generated a bovine drug (i.e., bovine CXCL8(3-74)K11R/G31P, bG31P) by mutating the first two amino acids at the beginning of the N-terminus of bovine CXCL8/IL-8 and later substituting Arg for Lys11 and Pro for Gly31. Bovine G31P was shown to be a highly effective ELR-CXC chemokine and neutrophil antagonist in cattle & guinea pigs, but a human equivalent thereof would be of significantly more use in human medicine. Published studies on the structure and function of human CXCL8 suggest that human CXCL8(3-72)K11R/G31P (i.e., hG31P) would not be a particularly effective chemokine antagonist. Thus, development of a humanized form of bG31P became a primary goal. I first examined the effect of wholesale ligation of the carboxy half of hCXCL8 onto the amino half of bG31P and generated a human-bovine chimeric G31P (hbG31P; i.e., bCXCL8(3-44)K11R/G31P-hCXCL8(45-72)). I also made substitutions at each remaining human-discrepant amino acid (i.e., T3K, H13Y, T15K, E35A, and S37T) within the 5 half of the hbG31P cDNA. The results showed that hbG31P and its analogues blocked CXCL8-induced human neutrophil chemotactic responses, reactive oxygen intermediate (ROI) release, and intracellular calcium flux. Humanized bovine G31P was also shown to significantly block pulmonary neutrophilic pathology in a guinea pig model of airway endotoxemia.<p> As bG31P, hbG31P and its further humanized forms showed essentially equivalent ELR-CXC chemokine antagonist activity, Dr. Fang Li, Ms Jennifer Town and I then generated a fully human form of bG31P, hG31P. <i>In vitro</i>, hG31P was shown to effectively inhibit CXCL-1-, -5-, and -8-induced neutrophil chemotactic responses, intracellular Ca2+ flux, and ROI release. Human G31P also desensitized heterologous G protein-coupled receptors (GPCR) including bacterial peptides (e.g., N-formyl-methionine-leucine-phenylalanine, fMLP), anaphylatoxin (e.g., complement 5a, C5a), lipid mediators (e.g., leukotriene B4, LTB4; platelet-activating factor, PAF) receptors. Moreover, hG31P, in a dose-dependent manner suppressed CXCL1 and CXCL8 expression by LPS-challenged airway epithelial cells and reversed the anti-apoptotic influence of ELR-CXC chemokines on neutrophils. <i>In vivo</i>, hG31P was significantly effective in blocking the pathology associated with airway endotoxemia, aspiration pneumonia, and intestinal ischemia and reperfusion injury, including neutrophil recruitment (70-95% reduction) into, and activation within, the airways or gut, chemokine or cytokine expression, and pulmonary vascular complications. The blockade of neutrophil recruitment by hG31P in aspiration pneumonia animals did not increase airway bacterial growth. The G31P treatment was protective in both mesenteric (i.e., local) and remote organ injury. These findings suggest that hG31P is not only a potent neutrophil antagonist, but an effective blocker of other inflammatory responses. These comprehensive anti-inflammatory effects indicate that hG31P could potentially provide a viable therapeutic approach for inflammatory diseases such as ALI /ARDS.

Rheumatoid arthritis is an autoimmune disease that can cause chronic inflammation of the joints and other areas of the body. Neutrophils contribute to the pathogenesis of arthritis, and are recruited to the site of inflammation by chemokines. CXCL8/IL-8 is a member of a sub-family of chemokines (ELR+CXC chemokines) that activate and attract neutrophils through the CXCR1 and CXCR2 receptors. Our lab developed a high affinity human CXCR1/CXCR2 antagonist, called human CXCL8(3-72)K11R/G31P (hG31P). This antagonist has been shown to be highly effective in blocking ELR+CXC chemokine-driven neutrophilic inflammation. In this study we looked at the therapeutic effect of blocking ELR+CXC chemokine receptors (CXCR1 and CXCR2) in an experimental model of arthritis. We induced type II collagen (CII)-induced arthritis (CIA) in mice and treated them with hG31P after the onset of disease. The parameters we looked at to assess disease severity were clinical scores (paws were graded on the severity of edema), clinical measurements (measuring inflammation by change in circumference of paw), serum levels of anti-CII antibodies, and inflammatory cytokines mRNA (IL-1β, TNF, KC, and MIP-2) and protein levels (IL-1β, IL-6, KC, and MIP-2) in paw tissue. Initially, when we analyzed all mice together, we were unable to see a change in clinical scores and measurement when CIA mice were treated with hG31P. All CIA mice did not develop arthritis simultaneously, but rather in a serendipitous fashion; therefore we subdivided our mice and analyzed data from mice that developed arthritis early versus those that developed it late. Treatment with hG31P in mice that developed arthritis early (within 5 weeks of initial CII injection) significantly reduced clinical scores (p=0.02) in one, but not both, of our experiments. When CIA mice were treated with hG31P we saw a significant reduction (p<0.05) in CII-specific IgG1 and MIP-2 protein levels in one of our experiments. Our results were variable and we did not see these changes in our other experiment. Treatment of CIA mice with G31P did not significantly affect inflammatory cytokine mRNA levels in the paws. During this study we found the production of anti-hG31P antibodies in our hG31P-treated mice. We used a Ca2+ influx assay to determine if these hG31P antibodies were neutralizing. When these antibodies were non-neutralizing we were able to see a significant reduction in the clinical scores (p=0.02) of our hG31P-treated CIA mice (that had developed early-onset arthritis) when compared to our saline-treated CIA mice. In the experiment in which we detected significant levels of neutralizing anti-hG31P antibodies, treatment with hG31P did not affect the clinical scores of our CIA mice. Although we cannot definitively say that hG31P has a therapeutic effect in CIA, we believe this line of research merits further investigation. Our research suggests to us that after some experimental refinement and reduction of the immune response mounted to hG31P, there could still be potential for hG31P to have a therapeutic effect in arthritis.

Thèse (M.Sc.)--Université Laval, 2007. / Bibliogr.: f. 80-90. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

CXCL16 is an interferon-inducible chemokine of the CXC-subfamily and functions as an adhesion molecule, when membrane bound, and a chemoattractant when soluble. Upregulation of cell associated CXCL16 (cCXCL16) in colorectal cancer is associated with increased tumor infiltrating lymphocytes and good prognosis. ADAM10 and ADAM17 are metalloproteinases responsible for cleaving CXCL16, releasing soluble CXCL16 (sCXCL16) and contributing to proliferation and migration of mesangial cells, in kidney inflammatory disease. We hypothesize that cCXCL16 is a substrate for ADAM10 and ADAM17 cleavage in colorectal cancer, releasing sCXCL16, which mediates cell proliferation. To this end, we first identified CXCL16 in the human colon carcinoma cell line, RKO, by immunohistochemistry. cCXCL16 was found in the membrane, cytoplasm and nucleus. We treated RKO, in vitro, with an inflammatory cytokine mix containing 1.4 nM rhIFN[gamma], 2.0 nM rhTNF[alpha] and 2.0 nM rhIL1[beta] to increase the cleavage of cCXCL16 to sCXCL16. Overnight incubation with the cytokine mix significantly (P=.004) increased the release of sCXCL16 compared to normal conditions. To confirm that a metalloproteinase is responsible for the cleavage of cCXCL16, we used a broad spectrum metalloproteinase inhibitor, GM6001, in combination with inflammatory stimulation, in cell culture. We assayed the supernatant using ELISA for sCXCL16. GM6001 at 100 [mu]M decreased sCXCL16 to levels indistinguishable from the background. Using siRNA, we knocked down the expression of ADAM10 and ADAM17, independently, to determine if the activity of each on cCXCL16 was mediated by inflammatory stimulation. It was shown that ADAM10 constitutively cleaved cCXCL16, and ADAM17 cleavage activity was induced by inflammatory stimulation. To determine if sCXCL16 increased colorectal cancer cell (CRC) proliferation through ligand-receptor binding, we treated cells with a range of rhCXCL16 from 3.125-100 ng/mL. rhCXCL16 did not increase RKO proliferation at doses up to 100 ng/mL. We used GM6001, to inhibit the cleavage of cCXCL16 into sCXCL16 then performed an ATPase assay and 6 day cell cycle analysis, under inflammatory stimulation. Increased cleavage of cCXCL16 induced by inflammatory stimulation with the cytokine mix containing 1.4 nM rhIFN[gamma], 2.0 nM rhTNF[alpha] and 2.0 nM rhIL1[beta], increased RKO proliferation and reduced apoptosis. We conclude that ADAM10 and ADAM17 cleavage of cCXCL16 to sCXCL16 is increased by ADAM17 activation with inflammatory stimulation. The cleavage of the extracellular portion from cCXCL16 is associated with increased proliferation and decreased apoptosis of colorectal cancer cells.

Les neutrophiles sont des cellules jouant un rôle dans la défense immunitaire contre une vaste gamme de microbes dont le Streptococcus pneumoniae, un agent étiologique important causant la pneumonie. Une hiérarchisation des récepteurs chimiotactiques retrouvés sur les neutrophiles nous indique que les peptides N-formylés (fMLP) sont parmi les facteurs chimioattractants les plus puissants. Nous avons donc comparé leur pouvoir chimiotactique avec celui des chimiokines CXC (MIP-2 et KC) lors d'une pneumonie à pneumocoque. Pour ce faire, nous avons utilisé des antagonistes connus du récepteur du fMLP et des anticorps neutralisants anti-chimiokines. À la suite de l'injection de ces produits à différentes lignées murines infectées par de faibles et fortes concentrations de plusieurs souches de pneumocoques au profil variable, nous avons déterminé que le fMLP produit par la bactérie et les chimiokines produites par l'hôte exercent tous deux un effet significatif sur la migration neutrophilique mais dépendent directement de la hiérarchisation de ces récepteurs de chimioattractants. / Polymorphonuclear neutrophils (PMNs) are known to play a major role in the clearance of Streptococcus pneumoniae from infected hosts. PMNs migrate under the influence of chemoattractant molecules at the site of inflammation. A hierarchical potency exists among known chemoattractants such as bacterial derived N-formylated peptides (fMLP) and host-derived chemokines, but was never investigated in pneumococal pneumonia. We infected mouse strains from different genetic backgrounds with low and high inocula of various strains of S. pneumoniae. Using fMLP receptor (FPR) antagonists and CXC chemokine blocking antibodies, we showed that PMN emigration relies upon such hierarchy in vivo, and that it is species- and inoculum-dependent, and is most likely linked to fMLP-induced cross-desensitization of other chemoattractant receptors including those for C5a and chemokines.

<p>The Ras family of proteins, composed of H, N, and KRas, function as small GTPases that act as "molecular switches" relaying signals from the cell membrane to the rest of the cell in a highly regulated manner. However, Ras signaling is aberrantly activated in a majority of human cancers either through an activating mutation or by activation or overexpression of upstream or downstream elements in the Ras pathway, endowing cells with many tumorigenic phenotypes. Ras is known to promote tumorigenesis through activation of cell intrinsic signaling including the Raf, PI3K, and RalGEF pathways. In regards to the latter, RalGEFs activate two other small GTPases, RalA and RalB. The role of these two proteins in Ras-mediated cancer was poorly understood. I thus assessed the requirement of RalA and RalB in tumor metastasis discovering that both proteins promote this critical step in cancer.</p><p>Ras does not, however, function solely by intrinsic cell signaling. Indeed, it was recently shown that oncogenic Ras signaling induces secretion of cytokines, a category of small molecules involved in cell to cell communication and inflammatory response. Moreover, the release of these cytokines was shown to promote tumorigenesis in an extrinsic fashion by increasing tumor vasculature, or angiogenesis. I noted that one of these cytokines hCXCL-8 (IL-8) belonged to the ELR+ CXC family of cytokines, suggesting that the entire family of ELR+ CXC cytokines may promote Ras driven tumorigenesis. Indeed, I found that expression of oncogenic Ras led to secretion of all ELR+ CXC chemokines in oncogenic Ras driven tumor cell lines, a mouse tumor, a human tumor, and were sometimes elevated in the serum of pancreatic cancer patients, the cancer most associated with oncogenic Ras mutations. Moreover, knockdown of one of these chemokines, hCXCL-1, in pancreatic cancer cells or genetic ablation of the receptor for these cytokines in mice, reduced Ras driven tumorigenesis. Taken together, these results suggest that oncogenic Ras also promotes tumorigenesis through a cell extrinsic pathway by secretion of ELR+ CXC chemokines.</p> / Dissertation

Uveal Melanoma is the most common primary intraocular tumor in adults. Despite the advances in numerous ophthalmic techniques leading to the increased accuracy of diagnosing this malignancy, the ten-year mortality rate for patients has remained unchanged at approximately fifty percent. Knowing this, further understanding of the specific steps that occur within the metastatic cascade of uveal melanoma is required. / Our laboratory utilizes five human uveal melanoma cell lines (92.1, SP6.5, MKT-BR, OCM-1, UW-1) of known proliferative, invasive, and metastatic potential. We used four methods to characterize the presence and roles of the chemotactic factors CXCL12, CXCL8, CXCL1 and HGF in these five cell lines. We also used a novel peptide inhibitor (TN14003) to block the biological action of CXCL12 on its receptor CXCR4. / With the results obtained from this thesis, we were able to establish the novel presence and importance of the four chosen factors for this malignancy. We were also able to display the positive effects TN14003 had on inhibiting uveal melanoma cell migration in vitro. This may lead to a future therapeutic target, which ultimately may delay or inhibit the metastatic process in uveal melanoma patients, improving the present unaffected ten-year mortality rate.