Dissertations / Theses on the topic 'Chemoattractants'

The effect of the chemoattractant bourgeonal on [Ca\(^{2+}\)]i and chemotaxis in human sperm was investigated. Burgeonal induced a dose-dependent, slowly-developing tonic elevation in [Ca\(^{2+}\)]i, The response was dependent on capacitation. In low-Ca\(^{2+}\) or EGTA-buffered saline the response to bourgeonal was inhibited. Pretreating spermatozoa with bis-phenol (20μM) to release stored Ca\(^{2+}\) did not alter the response. Thus bourgeonal acts primarily by inducing Ca\(^{2+}\) influx. Treatment of sperm with bourgeonal caused an increase in [cAMP]. When cells were pretreted with bourgeonal in low-Ca\(^{2+}\)saline, subsequent introduction of Ca\(^{2+}\) resulted in a single, large [Ca\(^{2+}\)]i transient in >75% of the cells, indicating that sudden influx of Ca\(^{2+}\) caused closure of the bourgeonal-sensitive Ca\(^{2+}\)- channel. This negative feedback was not modulated by IBMX (1mM) or dbcAMP (1mM), indicating that cAMP was not involved and that a direct action Ca\(^{2+}\) was more likely. Both Ni\(^{2+}\) (10μM) and La\(^{3+}\) (100μM) inhibited the action of bourgeonal on [Ca\(^{2+}\)]i, suggesting a possible role of CNG channels. Exposing sperm to a temporal bourgeonal gradient caused a series of transient [Ca\(^{2+}\)]i elevations in >20% of the cells. A gradient of progesterone (another characterised chemoattractant for human sperm) induced similar Ca\(^{2+}\) oscillations (in >20% of the cells), which increased in amplitude and frequency in response to the increasing progesterone concentration. Human spermatozoa responded chemotactically to a 1nM bourgeonal gradient, Chemotaxis was dependent on capacitation. The response was inhibited in low [Ca\(^{2+}\)]o but was unaltered by TMB-8 (an inhibitor of stored Ca\(^{2+}\) store release), thus showing a dependence on Ca\(^{2+}\) influx similar to the [Ca\(^{2+}\)]i signal.

Some plants release thousands of viable cells from root caps into the soil. These cells can be technically defined as Root Border Cells (BRD cells) and may play a role in the regulation of microbial populations in the rhizosphere. Chemoattractants released from pea (Pisam sativum) to Agrobacterium tumefaciens were characterized by using lectin and chemical analysis for heat-stability, size, and solubility. To understand the process of BRD cell release, a relationship was established between pectolytic enzyme activity and the release of pea BRD cells.

Eosinophils are characteristic infiltrating cells in asthmatic airways and may contribute to allergic and inflammatory diseases pathology through the ability of their products to produce chronic inflammatory and remodelling processes. One focus of efforts to define new therapeutic targets for chronic inflammatory diseases has been the study of mechanisms through which eosinophils are recruited to tissues and become activated, leading to secretion of reactive oxygen species, inflammatory mediators, cationic proteins and cytokines. The signalling pathways responsible for evoking migratory and secretory response in human eosinophils are incompletely understood.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2012.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 111-122).
Chemoattractant gradients play important roles in the normal function of immune system, from lymphocyte homeostasis to mounting efficient immune responses against infection. Improved fundamental knowledge about the role of chemoattractant gradients developed around single source cells in controlling chemotaxis of "receiving" cells would not only greatly advance our understanding of the basic mechanisms of cell chemotaxis but also would inform strategies for modulating chemoattractant gradients in therapeutic applications, such as adjuvant materials for vaccines and cancer immunotherapy recruiting immune cells of interest. In this thesis, we first applied mathematical modeling to understand the key characteristics of chemoattractant gradients secreted from single source cells at physiological rates. During the transport of chemoattractants, we considered the diffusion of soluble attractants, binding to matrix and degradation by proteolytic enzymes. From the calculated chemoattractant concentration gradients, we predicted the characteristics of attractant receptor engagement on responding cells, and estimated the maximum stimulation distance effectively triggering chemotaxis of responding cells based on the threshold for receptor engagement gradients, a difference of ~10 ligand-engaged receptors between the front and back of responding cells. This characteristic maximum stimulation distance is a function of multiple parameters including secretion rate of the source cell, diffusion constant of the chemoattractant, interaction with matrix, degradation or clearance of chemoattractant in the tissues, and the density of source cells. In addition, chemokine receptor desensitization induced by chemoattractants could shorten the maximum stimulation distance. We then developed Artificial Secreting Cells (ASCs) to mimic real chemoattractant secreting cells using cell-sized polysaccharide-based hydrogel microspheres releasing chemoattractant in a controlled manner. These alginate hydrogel microspheres, ~30 [mu]m in size, were crosslinked with Ca2+ between gluronic acid units on alginate backbones and provided a natural and bioactive environment for chemokines. The chemokines could be loaded into these alginate microspheres by soaking them in concentrated chemokine solutions and released in a reversible manner. This approach was shown as a general strategy for several chemokines, such as CCL21, CCL19, CXCL10 and CXCL12. The loading and release properties of individual chemokines were highly correlated with the average charge density on protein surface. We have also demonstrated that the controlled gradients created by ASCs were similar to the modeled gradients developed around single source cells. Further we used 3D collagen hydrogels embedded with ASCs as an in vitro model to investigate single human T-cell and dendritic cell migration dynamics to CCL21 and CCL19 chemokine gradients. Individual T-cells exhibited a binary response to isolated attractant sources, migrating highly directionally or ignoring the gradient completely; the fraction of responding cells correlated with chemokine receptor occupancy induced by the gradient. In sustained gradients eliciting low receptor desensitization, attracted T-cells or dendritic cells swarmed around isolated ASCs for hours. With increasing ASC density, overlapping gradients and high attractant concentrations caused a transition from local swarming to transient "hopping" of cells bead to bead. Thus, diverse migration responses observed in vivo may be determined by chemoattractant source density and secretion rate, which govern receptor occupancy patterns in nearby cells.
by Yana Wang.
Ph.D.

Antimicrobial peptides form an important aspect of the innate immune response of mammals. They function to kill invading microorganisms and in many cases activate other aspects of the innate and adaptive immune systems. One of the largest families of antimicrobial peptides is the defensins. In vertebrates, there are three subfamilies of defensins, termed α, β and θ based on the connectivity of the six conserved cysteines. The β-defensins, which are produced mainly by epithelial tissues and keratinocytes, came to wider interest when it was proposed that their loss of function might play a role in the pathogenesis of cystic fibrosis (CF) lung disease. Previous work has suggested that the airway surface liquid (ASL) of primary cultures of human airway epithelial cells possess salt-sensitive antibacterial activity and that this is impaired in CF individuals by elevated levels of sodium chloride. Further work has also suggested that the β-defensins secreted by the airway epithelia might comprise an important component of this salt-sensitive defence system. The aim of this project was 1) to characterise the salt-sensitive antibacterial activity of members of the human and murine β-defensin subfamily, 2) to analyse their activity as chemoattractants, 3) to establish a cell culture-based system for the production of β-defensins to allow for greater analysis of their range of activities and 4) to verify the validity of novel human β-defensins identified by bioinformatics techniques. In this thesis describes the characterisation of the salt-sensitive activities of synthetic human β-defensin 2 (DEFB2), mouse β-defensin2 (Defb2), and a novel β-defensin related 1, Defrl, which lacks the first of the canonical six cysteines are described against a range of CF-related pathogens. This work has concluded that a) DEFB2, Defb2 and Defrl display, to varying degrees, salt-sensitive antibacterial activity, b) The differences observed between the activities of the peptides may represent the evolution of species-specific profiles of antibacterial activity for specific defensins. c) That the loss of in Defrl of the first canonical cysteine does not result in loss of antibacterial activity and, most interestingly, Defrl also demonstrates activity against B. cenocepacia - a pathogen normally resistant to the activity of antimicrobials. Data presented in this thesis also suggests that synthetic Defb2 and Defrl show chemotactic activity to CD4+ T-lymphocytes and to immature dendritic cells. This work concludes that, like human β-defensins 1 and 2, the murine β-defensins, Defb2 and Defrl, can act as a bridge between the innate and adaptive immune systems. In this thesis, expression pattern of five novel human β-defensins in a range of human tissues is also analysed. Evidence is presented that they are all expressed at high levels in the testis and that two of these genes are expressed at much lower levels a variety of other tissues. These data suggest that the β-defensins are an expanding, and potentially quite large, subfamily of genes, many of which are yet to be characterised in terms of their expression profile and the antimicrobial and chemotactic activities.

Sepsis-induced diaphragmatic force loss and failure are associated with an increased exposure to proinflammatory mediators. The septic diaphragm has recently been reported to overexpresse chemokines, including the CC chemokine MCP-1 (monocyte chemoattractant protein-1). This thesis seeks to address the significance of MCP-1 overproduction in diaphragm proinflammatory mediator expression and skeletal muscle contractile function. Neutralization of endogenous MCP-1, produced following administration of LPS, decreased transcription of iNOS, IL-6, IL-1alpha, IL-1beta and MCP-1 in the diaphragm and prevented a decrease in diaphragm force production. Furthermore, exogenous MCP-1 stimulated IL-6 and MCP-1 transcription in primary diaphragm myotubes, and injection of MCP-1 in the healthy EDL muscle led to contractile weakness. Taken together, these results suggest that increased MCP-1 production in the septic diaphragm stimulates proinflammatory mediator production by diaphragm myocytes, contributing to the muscle's contractile dysfunction.

Systemic sclerosis is a multisystem connective tissue disease characterised by skin thickening and widespread, but variable, visceral fibrosis. The aetiopathogenesis is likely to involve immunological activation and microvascular dysfunction leading to excessive accumulation of extracellular matrix (ECM) with increased production of collagen type I in lesional tissues. This implies a dysregulated repair process probably as a consequence of aberrant crosstalk between fibroblasts and inflammatory cells. It has been proposed that a hierarchical cascade of soluble mediators in which initial induction of proinflammatory cytokines expressed by the inflammatory infiltrate may lead to expression of profibrotic mediators including TGFβ. A salient feature of the inflammatory response is directional migration of leucocytes into subendothelial tissues orchestrated by chemokines in a spatially and temporally-regulated multistep process. Work described in this thesis explores the expression of chemokine, monocyte chemoattractant protein-3 (MCP-3/CCL7) in SSc and in murine models for SSc: type 1 tight skin mouse (Tsk1) and a transgenic mouse strain (TβRIIΔk) in which there is fibroblast-directed disruption of TGFβ signalling. The hypothesis that crosstalk between MCP-3 and TGFβ may modulate the signalling response in the fibrotic microenvironment was also explored. Overexpression of MCP-3 was demonstrated on cDNA expression profiling and protein analysis of neonatal Tsk1 and TβRIIΔk fibroblasts. This was supported by immunohistochemical studies on dermal tissues. Similar upregulation dermal patterns of MCP-3 protein expression were observed in the early stage of diffuse cutaneous SSc. Activation of collagen reporter genes by MCP-3 in transgenic mouse fibroblasts and wildtype neonatal mouse fibroblasts harbouring proα2(Ι)collagen promoter reporter gene constructs is mediated via sequences within the proximal promoter and is partly dependent on TGFβ. This coinduction between the two factors in the fibrotic response is also demonstrated by activation of TGFβ signalling pathways by MCP-3 leading to type I collagen secretion. In addition, MCP-3 gene expression is stimulated by TGFβ. Comparison of downstream signalling pathways that regulate collagen gene activation by both cytokines confirms the central role of MAPK pathway activation in mediating the effects of both factors. An additive effect of these two agonists was demonstrated for key TGFβ-regulated genes on comparative microarray analysis. Overall, these results demonstrate that overexpression of MCP-3 is a key biochemical feature of early stage SSc and murine models of SSc, and suggest a novel role for this chemokine as a profibrotic mediator in addition to its role in regulating leucocyte recruitment. Furthermore, there is a potentially important interplay between MCP-3 and TGFβ in modulation of the signalling response in the fibrotic microenvironment.

The present research was carried out to investigate the chemoattraction activity of rheumatoid and non rheumatoid synovial fluids for human lymphocytes separated from peripheral blood, synovial tissue and synovial fluid. The phenotyping of locomotor cells in response to these fluids was also studied. Established procedures were used to separate lymphocytes from blood, synovial tissue and synovial fluid. The level of the chemotactic factors in the synovial fluid was measured by commercial and in-house developed methods. The inhibitory effect of anti-inflammatory drags on the lymphocyte locomotion was also studied. The chemoattractant activity of synovial fluid for human lymphocytes was investigated using the following methods: i. A Polarization assay which measures the shape change from spherical or round to a polarized shape (i.e from immotile to a motile shape) following stimulation with chemoattractants. ii. Collagen gel invasion, which measures the migration of lymphocytes into collagen gels- containing chemoattractants. Two methods were used for phenotyping the cells responding to the synovial fluids (I) APAAP which allowed the detection of lymphocyte surface markers in stained cytospin preparations, (ii) FACS which allowed the detection of cell surface markers of lymphocytes recovered from collagen gels after collagenase digestion. In addition methods used to measure the levels of chemotactic factors in the synovial fluid, were (I) Commercial single antibody sandwich ELISA kits (R&D) which measured IL-2, IL-8, MIP-1α and MCP-I, (ii) In-house developed multiple antibody sandwich ELISA which measured IL-15 in the fluids. The ability of synovial fluids from patients with rheumatoid (n=35) and other arthritides (n=18) to attract lymphocytes from peripheral blood of normal subjects, from rheumatoid synovia, and from joint fluids, was studied. The majority of synovial fluids from 29 rheumatoid arthritis patients were strongly attractive for blood lymphocytes which had been cultured overnight. Three out of five fluids from OA also attracted lymphocytes but to a lesser extent than RA fluids. In addition four of seven fluids from other inflammatory arthritides also gave high responses Rheumatoid synovial tissue lymphocytes responded to synovial fluids without a requirement for a period of culture. In contrast lymphocytes derived from rheumatoid and other synovial fluids were completely unresponsive to locomotor stimulants. Most of the responding cells from blood mononuclear cell fractions were T lymphocytes and the CD45RO isotype was attracted preferentially. Rheumatoid synovial fluids contained IL-8 , IL-15, MIP-1α and MCP-1 at levels in the nanogram range, sufficient to attract lymphocytes, but levels of IL-2 were too low to exert a chemoattractant effect. In contrast the levels of chemotactic factors in OA fluids were low and these fluids also showed less activity in attracting lymphocytes. The activity of the fluids could not be abolished by treatment with antibodies to IL-8, IL-2, MTP-1α, MCP-1 or IL-15 tested individually, but combinations of these antibodies inhibited most of the activity, suggesting that attraction of lymphocytes by the fluids is due to a combination of attractants. The accumulation of lymphocytes within the synovial fluids was not correlated with any single chemotactic factor mentioned above, suggesting that such accumulation is due to combined chemoattractants. In the present study it was also observed that neutrophils separated from normal blood gave a strong chemotactic response to the synovial fluids. In contrast neutrophils separated from the synovial fluid were immotile, suggesting that these cells had an intrinsic defect or that their locomotion was selectively blocked by synovial fluid chemotactic inhibitors. Moreover there was no correlation between IL-8 or levels of any other single cytokine and the accumulation of these cells in the fluids, indicating the possibility of multiple chemotactic factor involvement. The manipulation of the locomotion activity of lymphocytes in vitro in response to synovial fluid was studied using anti-inflammatory drugs. It was demonstrated that NSAIDs (including Aspirin, Ibuprofen and indomethacin), DMARDs (including gold, D-penicillamine and primaquine) and cytotoxic drugs including rapamycin and cyclophosphamide had no inhibitory effect on lymphocyte locomotion. On the other hand cyclosporin A and Glucocorticosteroids (including dexamethasone, prednisone and prenisolone) showed a significant inhibitory effect.

Idiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease of unknown aetiology and no effective therapy; therefore, new therapies targeting specific pro-fibrotic pathways are urgently required. The expression of monocyte chemoattractant protein-l (MCP-lL a profibrotic chemokine, is elevated in IPF and may contribute to its pathogenesis. The mechanism of MCP-l overexpression in IPF is unclear. Lung fibroblasts are considered the main effector cells since they express collagen and various pro-fibrotic cytokines and chemokines that mediate lung fibrosis. Moreover, our group has recently reported promoter-specific epigenetic abnormalities to be responsible for targeted repression of two key anti-fibrotic genes in lung fibroblasts isolated from patients with IPF (F-IPF) compared to fibroblasts from non-fibrotic lungs (F-NL). Therefore, we hypothesize that constitutive and cytokine-induced MCP-l expression is up-regulated in F-IPF in comparison to F-NL, as a result of aberrant epigenetic modifications at the MCP-l gene promoter and enhancer, favouring transcription. Here we characterise the molecular mechanisms involved in increased expression of MCP-l in F-IPF. F-IPF and F-NL cells were provided by Dr Feghali-Bostwick (University of Pittsburgh, USA). MCP-l protein and mRNA expression were measured by ELlSA and quantitative RT-PCR (qRT-PCRL respectively. Cytokine receptor and transcription factor (TF) expression was analysed by Western blotting. Reporter gene assays were conducted to assess TF activation and involvement in MCP-l gene transcription in F-IPF and F-NL cells. Native TF binding and chromatin modifications were analysed via chromatin immunoprecipitation (ChiP) assays. MCP-l protein and mRNA levels were significantly higher in F-IPF compared with F-NL, both constitutively and in response to cytokine stimulation, and this overexpression was independent of increased mRNA stability or increased TF activation and expression in F-IPF. However, ChiP assays revealed increased basal and cytokine-induced TF binding at the MCP- 1 promoter and enhancer in native chromatin environment in F-IPF compared with F-NL. This was accompanied by increased histone H3 and H4 acetylation and increased recruitment of histone acetyltransferases (HAT) p300 and CBP to the promoter and enhancer. Inhibiting p300 with the HAT inhibitor LTK-14 decreased MCP-l protein expression in F-IPF cells to the levels observed in F-NL. Aberrant histone hyperacetylation at the MCP-l promoter and enhancer due to increased HAT recruitment plays a key role for increased TF binding and subsequent transcription of MCP-l in F-IPF cells. Our findings suggest that epigenetic abnormalities are involved in the overexpression of pro-fibrotic genes in IPF and therefore could become novel therapeutic targets. t r ABSTRACT Idiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease of unknown aetiology and no effective therapy; therefore, new therapies targeting specific pro-fibrotic pathways are urgently required. The expression of monocyte chemoattractant protein-i (MCP-i), a profibrotic chemokine, is elevated in IPF and may contribute to its pathogenesis. The mechanism of MCP-i overexpression in IPF is unclear. Lung fibroblasts are considered the main effector cells since they express collagen and various pro-fibrotic cytokines and chemokines that mediate lung fibrosis. Moreover, our group has recently reported promoter-specific epigenetic abnormalities to be responsible for targeted repression of two key anti-fibrotic genes in lung fibroblasts isolated from patients with IPF (F-IPF) compared to fibroblasts from non-fibrotic lungs (F-NL). Therefore, we hypothesize that constitutive and cytokine-induced MCP-i expression is up-regulated in F-IPF in comparison to F-NL, as a result of aberrant epigenetic modifications at the MCP-i gene promoter and enhancer, favouring transcription. Here we characterise the molecular mechanisms involved in increased expression of MCP-i in F-IPF. F-IPF and F-NL cells were provided by Dr Feghali-Bostwick (University of Pittsburgh, USA). MCP-i protein and mRNA expression were measured by ELlSA and quantitative RT-PCR (qRT-PCR), respectively. Cytokine receptor and transcription factor (TF) expression was analysed by Western blotting. Reporter gene assays were conducted to assess TF activation and involvement in MCP-i gene transcription in F-IPF and F-NL cells. Native TF binding and chromatin modifications were analysed via chromatin immunoprecipitation (ChiP) assays . . MCP-i protein and mRNA levels were significantly higher in F-IPF compared with F-NL, both constitutively and in response to cytokine stimulation, and this overexpression was independent of increased mRNA stability or increased TF activation and expression in F-IPF. However, ChiP assays revealed increased basal and cytokine-induced TF binding at the MCP- 1 promoter and enhancer in native chromatin environment in F-IPF compared with F-NL. This was accompanied by increased histone H3 and H4 acetylation and increased recruitment of histone acetyltransferases (HAT) p300 and CBP to the promoter and enhancer. Inhibiting p300 with the HAT inhibitor LTK-14 decreased MCP-i protein expression in F-IPF cells to the levels observed in F-NL. Aberrant histone hyperacetylation at the MCP-i promoter and enhancer due to increased HAT recruitment plays a key role for increased TF binding and subsequent transcription of MCP-i in F-IPF cells. Our findings suggest that epigenetic abnormalities are involved in the overexpression of pro-fibrotic genes in IPF and therefore could become novel therapeutic targets.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
Novos biomarcadores da funÃÃo renal estÃo sendo estudados com o propÃsito de detectar precocemente alteraÃÃes renais em portadores de AF, dentre eles encontra-se a proteÃna quimiotÃtica de monÃcitos 1 (MCP-1), uma quimiocina de monÃcitos e macrÃfagos, produzida por cÃlulas do sistema renal em resposta ao processo de isquemia-reperfusÃo. OBJETIVO: Avaliar o uso de MCP-1 como biomarcador de lesÃo renal precoce em pacientes adultos com anemia falciforme em uso ou nÃo de hidroxiureia (HU). METODOLOGIA: Participaram do estudo 50 pacientes: 30 em uso de (HU)-grupo SSHU e 20 sem HU-grupo SS. Um grupo controle foi composto por 20 indivÃduos com HbAA, sem complicaÃÃes renais. ProteinÃria, albuminÃria, creatinina e urÃia urinÃrias, marcadores do estresse oxidativo como MDA e NOx foram determinados por mÃtodos espectrofotomÃtricos. MCP-1 urinÃrio foi detectado por enzima imunoensaio (ELISA). Os dados clÃnicos e de hemograma, creatinina e ureia sÃricas foram retirados do prontuÃrio mÃdico. Foi coletada a primeira urina do dia. O programa Graph Pad Prism 5.0 foi utilizado para anÃlise estatÃstica. A comparaÃÃo das mÃdias entre os grupos foi realizada atravÃs do teste t de Student e anÃlise de variÃncia (ANOVA). RESULTADOS E DISCUSSÃO: Albumina urinÃria esteve maior nos pacientes em relaÃÃo ao grupo controle (Controle-3.12 Â 4.35; SSHU- 11.85 Â 9.16; SS- 14.13 Â 12.22; p <0.0001). A taxa de filtraÃÃo glomerular estimada apresentou-se significantemente menor no grupo controle (Controle- 95.9 Â 19.92; SSHU- 137.9 Â 40.7 e SS- 140.1 Â 53.9; p= 0.0024). Observaram-se nÃveis elevados de MCP-1 (Controle- 42.12 Â 27.6; grupo SSHU- 166.2 Â 88.37 e grupo SS- 219.7 Â 115.0; p<0.001; p=0.039); MDA (Controle- 2.29 Â 1.13; grupo SSHU-5.25 Â 2.33 e grupo SS- 6.93 Â 2.12; p<0.0001;p=0.006) e NOx (Controle-2.25Â1.9; grupo SSHU-56.54 Â 9.15 e grupo SS 39.12 Â9.02; p<0.0001; p=0.001) nos pacientes em comparaÃÃo aos controles saudÃveis, e mais elevados no grupo SS em relaÃÃo ao grupo SSHU. Os pacientes com haplÃtipo Bantu/Bantu apresentaram maior concentraÃÃo de MCP-1, independente do uso de HU, seguido de Bantu/ Benin e Benin/Benin (p=0.01). Observou-se correlaÃÃo positiva entre os uma correlaÃÃo entre os nÃveis de MCP-1 e contagem de monÃcitos (p=0.004; r= 0.42); proteinÃria (p=0.002; r=0.43); albuminÃria (p=0.0004; r=0.47); TFG (p=0.02; r=0.32); MDA (p=0.02; r=0.32) e NOx (p=0.007; r= 0.38). CONCLUSÃO: Os resultados indicam que MCP-1 foi preditivo na detecÃÃo de alteraÃÃo renal, e que pode estar correlacionado ao dano causado pelo estresso oxidativo nos rins, evidenciado pelos altos nÃveis de MDA. Ainda, a HU parece ter reduzido o dano renal, visto que os pacientes em uso do fÃrmaco apresentaram nÃveis reduzidos desses parÃmetros.

Efficient directed migration requires tight regulation of chemoattractant signal transduction pathways in both space and time, but the mechanisms involved in such regulation are not well understood. Here, we investigated the role of protein kinase A (PKA) in controlling signaling of the chemoattractant cAMP in Dictyostelium discoideum. We found that cells lacking PKA display severe chemotaxis defects, including impaired directional sensing. Although PKA is an important regulator of developmental gene expression, including the cAMP receptor cAR1, our studies using exogenously expressed cAR1 in cells lacking PKA, cells lacking adenylyl cyclase A (ACA) and cells treated with the PKA-selective pharmacological inhibitor H89, suggest that PKA controls chemoattractant signal transduction, in part, through the regulation of RasG, Rap1 and TORC2. As these pathways control the ACA-mediated production of intracellular cAMP, they lie upstream of PKA in this chemoattractant signaling network. Consequently, we propose that the PKA-mediated regulation of the upstream RasG, Rap1 and TORC2 signaling pathways is part of a negative feedback mechanism controlling chemoattractant signal transduction during Dictyostelium chemotaxis.

Chemotaxis is a fundamental process involved in a diverse range of biological phenomena such as nutrient finding in unicellular organisms, development and morphogenesis of multicellular organisms, wound healing and immune response. It is also related to a number of diseases including immunological disorders and cancer metastasis. It is a complex cellular process which is composed of several steps including signal detection and amplification, cell polarisation and cytoskeleton reorganisation. There are several signal transduction pathways working in parallel to control chemotaxis, which involve a multitude of regulatory factors and effectors modulating the cytoskeletal dynamics. The final outcome is actin polymerisation at the leading edge, depolymerisation at the trailing edge and myosin II filament assembly and contraction in the back of the cell, which together drive the directional cell movement in the direction of the chemotactic gradient. One of the poorly understood aspects of the chemotactic response is a biphasic character of the chemoattractant-induced actin polymerisation. The two temporally distinct phases of actin polymerisation reveal very different characteristics and regulation, but the exact mechanisms responsible for these differences have not been determined. We have taken advantage of recent advances in a quantitative proteomic technique and adapted the SILAC experimental approach to analyse the fast translocation dynamics of proteins associated with a crude cytoskeleton preparation after stimulation with the chemoattractant in Dictyostelium discoideum. These experiments detected, among many other proteins, a wide range of cytoskeletal components including structural constituents, actin binding proteins and regulatory factors associated with the cortical cytoskeleton. The SILAC results provide valuable quantitative information about the translocation dynamics for hundreds of cytoskeletal components in the same experiment. Most of those proteins follow the generic biphasic profile of enrichment reflecting actin dynamics, but subsets of proteins show specific incorporation during only one of the two phases of actin polymerisation. We identify several positive regulators of actin polymerisation showing enrichment specific to the first phase of chemotactic response and other regulatory components incorporated to the cytoskeleton exclusively during the second phase of cAMP-induced actin polymerisation. Detailed analysis of the major structural components of the actin cytoskeleton reveal that about 25% of the primary actin nucleator – Arp2/3 complex, incorporated to the cortex during the first phase, do not trigger formation of a new filament. In order to verify the proteomic analysis 55 known cytoskeletal proteins, which showed strong enrichment in the SILAC experiments, were selected for tagging with GFP followed by in vivo fluorescence microscopy imaging. Their analyses served as positive controls validating the translocation dynamics patterns measured with the SILAC experiments and also provided further detailed spatio-temporal characterisation of the cAMP-mediated protein translocation. Regulatory factors involved in the first phase of actin polymerisation can be classified into two groups exhibiting distinct incorporation dynamics detected by TIRFM imaging. The first group includes DocA, DocB and GacR proteins, which show very rapid and transient translocation to the cortex and are most likely involved in the earliest events following cAMP stimulation. The second group, which is composed of Roco7, PakB, XacB and GacA proteins, shows slower enrichment and longer occupancy at the cortex. TIRFM analysis also led to characterisation of a new class of cytoskeletal components, which bind exclusively to the pre-existing actin filaments and do not associate with the structures formed during response to cAMP stimulation. These proteins, which include MhcA, ZizA, RapGAP1 and two novel cytoskeletal components, translocate only to the inner cortex upon cAMP stimulation and are likely involved in regulation of the actin depolymerisation phase. In order to identify novel components of the cytoskeleton 54 uncharacterised proteins showing response to cAMP stimulation in the SILAC experiments, were cloned and tagged with GFP. Most of those proteins showed specific localisations to various cellular compartments including the nucleus, contractile vacuoles and various types of vesicles. 12 of the selected proteins showed colocalisation with the cortical structures and clear translocation in response to cAMP stimulation. A small number of these proteins contain actin binding domains identified by sequence homology analysis, but none of them were previously characterised. These novel cytoskeletal components need to be further investigated in order to determine their functions in the cytoskeleton and chemotaxis.

Sponges are the descendants of the oldest members of the metazoan phylogenetic lineage and their genome contains animal specific genes but lack true tissues, organ systems, and neurons. Thus, the sponge model system can be used to elucidate origin of developmental processes. The PSED (RDGN) network (Pax/Six/Eya/Dac) is important in development of eyes, muscles, and other structures in Bilaterians. Similarly, sponges contain a precursor Pax-Six gene network. The Ephydatia muelleri (Em) PaxB protein binds to a Pax2/5/8 consensus sequence site and two cis-regulatory elements upstream and one intron sequence of EmSix1/2 (Rivera et al., 2013). This study aimed to determine if transcription factor EmSix1/2 binds upstream of EmPaxB using gel shift mobility assays, identify other downstream targets of EmSix1/2 using DNA immunoprecipitation, and to identify the recognition sequence of Six in sponges through sequencing. In conclusion, purified EmSix binds to DNA specific fragments (1 and 3), which may contain enhancer sequences located in the PaxB promoter region.Possible consensus recognition sequence of Six in sponges were also identified. The host immune response has various mechanisms to protect the organism from infections and invasions of microorganisms and cell damaging chemicals. One such mechanism is the elimination of reactive oxygen species aided by superoxide dismutase (SOD). A study showed that anti-neutrophil chemotactic factor antibodies recognize Tritrichomonas foetus SOD (Granger et al., 1997). During first casualties, SOD is released and triggers a host immune response. The parasites could use SOD to counter oxidative attacks by the leukocytes and damaged cells to protect surrounding parasites. We used the parasite Trichomonas vaginalis that causes trichomoniasis to determine if SOD acts a neutrophil (or other leukocyte) chemotactic factor in this parasite and characterize the expression and secretions of epithelial cells when treated with SOD6. HeLa cells treated with SOD6 showed an increase in the expression of IL-8 chemokine relative to TrxR treated cells. Scratch test assays showed that SOD may act as a macrophage chemoattractant as compared to TrxR but further tests are needed to confirm this.

Thesis (MSc)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: Bacterial chemotaxis, as observed for Escherichia coli, in a field of chemoattractant molecules is characterised by a run-and-tumble motion. The motion is effected by the clockwise (CW) or counter-clockwise (CCW) rotation of flagella; filamentous appendages attached to molecular motors on the cell body. Runs appear when all flagella turn in the CCW-direction and are used to maintain a favourable direction. Tumbles emerge as soon as one flagellum starts to turn CW and are used for reorientation. Because of the variation observed between individual bacteria displaying run-and-tumble motion, we choose to model this behaviour within a probabilistic framework. An important feature of the chemotactic ability of E.coli is that the cell increases run while moving in the right direction and shortens it in the opposite case. This underlines that tumbles are used for reorientation. It has been found from experiments that there can be significant variation in the tumble fashion depending on the fraction of CW-rotating motors (Turner et al., 2000). The change in angle produced when fewer flagella are rotating CW was found to be smaller when compared to the case for many CW-rotating flagella. In addition, the change of direction contributed by a small portion of CW-rotating flagella is rarely significant for bacteria with many flagella. Based on these observations, we have distinguished between models for the one-flagellated and the multi-flagellated cases. Furthermore, since the tumbling angle change increases with the fraction of CW-rotating motors, it would not be impossible to have some cases where the amount of turn produced by the CW-rotating motors induces the bacterium to have a change of direction greater than 2π. But, this feature could not have been observed because when the bacterium tumbles it can effectuate several revolutions before resuming to a new direction. Therefore, we do not restrict our change of direction to (0,2π) to allow the bacteria to have the possibility to effectuate change of directions of magnitude greater than 2π. To this end, we differentiate between the probability of having directional change of magnitude α and α +2π . Thus we do not use angle change distributions that are defined modulo 2π such as the von Mises distribution or the wrapped normal distribution. The chemotactic ability of the bacterium is modelled by representing the CCW-bias of a single flagellum as a function of the chemoattractant concentration. The model includes the temporal memory of chemoattractant concentration that the bacterium has, which usually spans about 4s. The information about the quality of the current direction of the bacterium is transmitted to the flagellar motor by assuming that this one varies with the chemoattractant concentration level. In addition, the saturation of the bias is incorporated by assuming that the bacterium performs a temporal comparison of the receptor occupancy. The present CCW-bias-Model accounts for the chemotactic ability of the bacterium as well as its adaptation to uniform chemoattractant environment. The models of one-flagellated and multi-flagellated bacterial motion, are used to investigate two main problems. The first one consists of determining the optimal tumbling angle strategy of the bacteria. The second one consists of looking at the effects of the tumble variation on the chemotactic efficiency of the bacteria. In order to address these questions, the chemotactic efficiency measure is defined in such a way that it reflects the ability of the bacteria to converge and to stay in a near neighbourhood of the source so that they gain more nutrients. Since its movement is entirely governed by its single flagellum, the one flagellated bacterium is more able to effectuate a run motion. Tumbling events are modelled to be all equivalent because there is not any fraction of flagella to consider. On the other hand, the tumble variation of the multi-flagellated bacteria is modelled by assuming that the directional change during a tumble is a function of the fraction of CW-rotating motors. By assuming that the number of CW-rotating flagella follows a binomial distribution, we suppose that the multi-flagellated bacteria are less able to effectuate a run motion. This also implies that the change of direction produced by fewer CW-rotating flagella are more likely to happen, and this compensates the lack of run. The models show that the optimal tumbling angle change for the bacteria is less than 2π and that higher flagellated bacteria have higher chemotacitc efficiency. As the number of flagella of the bacteria increases, there can be more tumble variation, in this case the bacteria are more capable of adjusting their direction. There could be some situation were the bacteria are not moving to the right direction, but do not require a large change of direction. This ability to adjust their direction accordingly allows them to converge nearer to the source and to gain more nutrients. In addition, the dependence of the tumbling angle on the fraction of CW-rotating flagella of the mutli-flagellated bacteria, implies that there is a correlation between the tumbling angle deviation and the external environment, because the rotational states CCW-CW of the flagella depends on the external cue. Consequently, it would not be impossible that the average magnitude of tumbling angle change depends on the external environment. To investigate this possibility we analyse the distribution of the tumbling tendency of a single bacterium over time, which is the distribution over time of the average positive tumbling change of the bacterium, within zerogradient environment and within non-zero-gradient environment. We defined the average of these tumbling tendency over time as the directional persistence. We observe that the directional persistence within these different nonzero- gradient environment remains the same. However, the difference between the directional persistence within zero-gradient and non-zeros gradient environment gets larger as the number of flagella of the cell increases. There is more correlation between the external environment and the tumbling tendency of the bacterium. Which is the reason why the higher flagellated bacteria responds the best to the external environment by having the higher chemotactic performance. Finally, the total directional persistence generated by the optimal tumbling angle change of the bacteria is the average directional persistence of the bacteria regardless of their number of flagella. Its value, predicted by the model is 1.54 rad within a non-zero-gradient environment and 1.63 rad within a zero-gradient environment.
AFRIKAANSE OPSOMMING: Bakteriese chemotakse, soos waargeneem word vir Escherichia coli, in ’n veld van chemiese lokmiddel molekules word gekenmerk deur ’n hardloopen- tuimel beweging. Die beweging word bewerkstellig deur die regsom of linksom rotasie van flagella; filamentagtige aanhangsels geheg aan molekulêre motors op die selliggaam. ’n Hardloop aksie kom voor as al die flagella linksom roteer en word gebruik om ’m voordelige koers te handhaaf. Tuimels kom voor sodra een van die flagella regsom draai en word gebruik vir heroriënteering. Van wee die variasie wat waargeneem word tussen individuele bakterieë wat hardloop-en-tuimel bewegiging vertoon, verkies ons ’n probabilistiese raamwerk om in te werk. ’n Belangrike eienskap van die chemotakse vermoë van E. coli is dat die sel meer gereeld hardloop terwyl dit in die regte rigting beweeg en minder gereeld in die teenoorgestelde geval. Dit beklemtoon dat tuimels gebruik word vir heroriënteering. Dit is al eksperimenteel vasgestel dat daar betekenisvolle variasie kan wees in die tuimel wyse, wat afhang van die breukdeel regsom roterende motors (Turner et al., 2000). Die hoekverskil afkomstig van minder regsom roterende flagella was vasgestel om kleiner te wees in vergelyking met die menig regsom roterende geval. Verder word die bydrae tot die hoekverskil van ’n klein breukdeel regsom roterende flagella selde beduidend vir bakterieë met baie flagella. As gevolg van hierdie waarnemings, tref ons onderskeid tussen modelle vir een-flagella en multiflagella gevalle. Aangesien die tuimel hoeksverskil vergroot saam met die breukdeel regsom roterende motore, is dit nie onmoontlik om gevalle te hê waar die hoeveelheid draaiaksie gegenereer deur die regsom roterende motore ’n rigtingsverskil groter as 2π kan bewerkstellig nie. Dit was nie moontlik om hierdie eienskap waar te neem nie aangesien die bakterieë ’n paar keer kan tuimel voordat ’n nuwe rigting vasgestel word. Vir hierdie rede beperk ons nie die hoeksverskil tot (0,2π) nie om die bakterieë toe te laat om rigtings veranderinge groter as 2π te ondergaan. Vir hierdie doel, onderskei ons tussen die waarskynlikheid van ’n rigtinsverskil met grootte α en α + 2π. Dus, gebruik ons nie hoekverskil verspreidings wat modulo 2 gedefinieer is nie, soos die von Mises verspreiding of omwinde normaalverdeling. Die chemotakse vermoë van die bakterium word gemodelleer deur die linksom sydigheid van ’n enkele flagellum as ’n funksie van die chemotakse lokmiddel konsentrasie voor te stel. Die model sluit in die tydelike geheue wat die bakterium besit oor chemotakse lokmiddel konsentrasie, wat gewoonlik oor 4s strek. Die informasie oor die kwaliteit van die huidige rigting van die bakterium word deur gegee na die flagella motor toe deur die aanname te maak dat dit wissel met die chemotakse lokmiddel konsentrasie vlak. Die versadiging van die sydigheid word geinkorporeer deur aan te neem dat die bakterium ’n temporale vergelyking maak tussen reseptor okkupasie. Die huidige linksom sydige model neem die bakterium chemotakse vermoë in ag, as ook aanpassing tot ’n uniforme chemotakse lokmiddel omgewing. Die modelle van een-flagella en multi-flagella bakteriële beweging word gebruik om twee hoof probleme te bestudeer. Die eerste, bestaan daaruit om vas te stel wat die optimale tuimel hoek strategie van die bakterieë is. Die tweede kyk na die uitwerking van tuimel variasie op chemotakse effektiwiteit. In orde om hierdie vra te adreseer word die chemotakse effektiwiteit op so mannier gedefinieer dat dit die bakteriese vermoë om die buurt om die oorsprong te nader en daar te bly. Aangesien die beweging heeltemal vasgestel word deur een flagella, in die een-flagella geval, is ’n bakterium meer in staat daartoe om ’n hardloop aksie te bewerkstellig. Tuimel voorvalle word as ekwivalent gemodeleer omdat daar geen breukdeel roterende flagella is om in ag te neem nie. In teenstelling, word die tuimel variasie van multi-flagella bakterieë gemodeleer deur die aanname te maak dat rigtingsverandering gedurende ’n tuimel ’n funksie is van die breukdeel regsom roterende motore. Deur die aanname te maak dat die getal regsom roterende flagella ’n binomiese verspreiding volg, veronderstel ons dat multi-flagella bakterieë minder in staat daartoe is om ’n hardloop aksie te onderneem. Hierdie impliseer ook dat rigtingverandering wat geproduseer word deur minder regsom roterende flagella meer geneig is om voor te kom en dan kompenseer vir ’n tekortkoming aan hardloop gebeure. Die modelle wys dat die optimale tuimelhoek verandering minder as 2 is en dat bakterieë met meer flagella meer chemotaksies effektief is. Soos die getal flagella vermeder, kan daar meer tuimel variasie wees, en in die geval is die bakterieë meer in staat om hul rigting te verander. Daar kan omstandighede wees waar die bakterieë nie in die regtige rigting beweeg nie, maar nie ’n groot rigtingsverskil nodig het nie. Hierdie vermoë om hul rigting byvolglik te verander stel hul in staat om nader aan die oorsprong te konvergeer en dus meer voedingstowwe op te neem. Die afhanklikheid van die tuimel hoek op die breukdeel regsom roterende flagella van multi-flagella bakterieë dui daarop dat daar ’n korrelasie is tussen die tuimel hoek afwyking en die eksterne omgewing, omdat die roterings toestand, regs- of linksom, van die flagella afhanklik is van die eksterne sein. As ’n gevolg, is dit nie onmoontlik dat die gemiddelde grootte van die tuimel hoek verandering van die eksterne omgewing afhang nie. Om hierdie moontlikheid te bestudeer, analiseer ons die verspreiding van die tuimel neiging van ’n enkele bakterium oor tyd, wat die verspreiding oor tyd van die gemiddelde positiewe tuimel verandering is, in ’n nulgradient en nie-nul-gradient omgewing. Ons het hierdie gemiddelde tuimel neigings oor tyd gedefinieer as die rigtings volharding. Ons het waargeneem dat die rigtings volharding binne verskillende nienul- gradient omgewings dieselfde bly. Nogtans is die verskil tussen die rigtings volharding binne nul-gradient en nie-nul-gradient omgewings groter soos die getal flagella vermeder. Daar is meer korrelasie tussen die eksterne omgewing en tuimel neiging van die bakterium. Dit is die rede hoekom bakterieë met meer flagella die beste reageer op die eksterne omgewing deur beter chemotakse effektiwiteit. Ten slotte, die totale rigtings volharding gegenereer deur die optimale tuimel hoek verandering is die gemiddelde rigtings volharding ongeag van die getal flagella. Die waarde wat deur die model voorspel word is 1.54 rad binne ’n nie-nul-gradient omgewing en 1.63 rad binne ’n nul-gradient omgewing.

Cardiovascular diseases are the number one cause of death in Western societies. Endothelial dysfunction is an early event in the pathology of atherosclerosis, which is an underlying cause in the majority of cardiovascular events. Exposure to persistent environmental pollutants, such as polychlorinated biphenyls (PCBs), is a risk factor for the development of atherosclerosis. First, we tested a hypothesis that coplanar PCBs, dioxin-like chemicals with affinity for aryl hydrocarbon receptor (AhR), can stimulate up-regulation of monocyte chemoattractant protein-1 (MCP-1), an endothelium-derived chemokine that attracts monocytes into sub-endothelial space in early stages of atherosclerosis. Coplanar PCBs 77 and 126 increased expression of MCP-1 in endothelial cells, and this effect was dependent on activation of AhR and increased levels of cytochrome P450 monoxygenases. Subsequent rise in the levels of reactive oxygen species (ROS) led to a downstream stimulation of redox-sensitive kinases and transcription factors. Lipid rafts, and particularly caveolae, are enriched in endothelial cells, and down-regulation of caveolin-1, a key structural protein of caveolae, decreases the progression of atherosclerosis. Studies using deletion of caveolin-1 in vitro and in vivo demonstrated that intact caveolae were required for up-regulation of MCP-1 and pro-inflammatory interleukin-6 (IL-6) by PCB77. Nutrition can modulate adverse outcomes of human exposure to environmental chemicals. Fish oil-derived long-chain omega-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA, 22:6ω-3), can alleviate inflammatory responses and the risk of cardiovascular disease. Cyclopentenone metabolites produced by oxidation of DHA contribute to these protective effects. Endothelial cells were pre-treated with oxidized DHA (oxDHA), prepared by incubation of the fatty acid with a free radical generator. Subsequent up-regulation of MCP-1 by coplanar PCB77 was markedly reduced. DHA-derived cyclopentenones increased nuclear translocation and DNA binding of a transcription factor NF-E2-related factor-2 (Nrf2), as well as expression levels of its target, antioxidant enzyme NAD(P)H:quinone oxidoreductase (NQO1). This stimulation of antioxidant responses prevented ROS production and inflammatory responses induced by PCB77. These data support the concept that nutrition prevents toxicity caused by environmental pollutants; thus, nutrition and can be a sensible approach to alleviate chronic pathologies associated with these chemicals.

Thromboxane A2 receptor (TP) has been shown to play important roles in multiple aspects of cancer development including regulation of tumor growth, survival and metastasis. Molecular mechanisms of TP mediated cancer cell invasion remain to be identified. TP agonist, I-BOP, significantly elevated several matrix metalloproteinases (MMPs) including MMP-1, MMP-3, MMP-9 and MMP-10 in A549 human lung adenocarcinoma cells overexpressing TPα (A549-TPα) or TPβ (A549-TPβ). Signaling pathways of I-BOP-induced MMP-1 expression were examined in further detail as a model system for MMPs induction. Signaling molecules involved in I-BOP-induced MMP-1 expression were identified by using specific inhibitors including small interfering (si)-RNAs of signaling molecules and promoter reporter assay. The results indicate that I-BOP-induced MMP-1 expression is mediated by protein kinase C (PKC), extracellular signal-regulated kinase (ERK)-activator protein-1(AP-1) and ERK-CCAAT/enhancer-binding protein β (C/EBPβ) pathways. I-BOP-induced cellular invasiveness of A549-TPα cells was blocked by, GM6001, a general inhibitor of MMPs. Knockdown of MMP-1 and MMP-9 by their respective siRNA partially reduced I-BOP-stimulated A549-TPα cells invasion suggesting that other MMPs induced by I-BOP were also involved. Furthermore, secreted MMP-1 in conditioned media from I-BOP-treated A549-TPα cells (CM-I-BOP) autocrinely induced monocyte chemoattractant protein-1 (MCP-1) expression. The induction of MCP-1 by MMP-1 in A549 cells was via activation of protease-activated receptor 2 (PAR2) instead of commonly assumed PAR1. This conclusion was reached from the following findings: (1) expression of MCP-1 induced by trypsin, a PAR2 agonist, was inhibited by a PAR2 antagonist. (2) expression of MCP-1 induced by MMP-1 and by CM-I-BOP was blocked by a PAR2 antagonist but not by other PAR antagonists; (3) expression of MCP-1 induced by MMP-1 and by CM-I-BOP was attenuated significantly by pretreatment of cells with PAR2-siRNA. Finally, MCP-1 also can be induced by direct activation of TP in a SP1 involved mechanism. CM-I-BOP enhanced MCP-1-dependent migration of RAW 264.7 macrophages. Co-culture of A549 cells with RAW 264.7 macrophages induced expression of MMPs, VEGF and MCP-1 genes, and increased the invasive potential in A549 cells. My studies provide molecular mechanisms by which TP-mediated cancer cell invasion and suggest that TP is a potential anti-cancer drug target.

Traumatic Brain Injury (TBI) is one of the leading causes of death and disability among young adults and has been a significant field in medical research over the past decades. Intensive studies focusing on how to repair tissue damage resulting from head injuries have discovered that the central nervous system (CNS) retains a regenerative capacity throughout life due to the persistent presence of neural stem/progenitor cells (NS/NPCs) in the neurogenic regions. In the normal brain, cells generated in the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to the olfactory bulb and cells in the subgranular zone (SGZ) migrate laterally into the granule cell layer of the dentate gyrus. Directed movement of these NS/NPCs is controlled by a variety of factors, and among them the chemoattractant SDF-1 is of particular importance. Studies have identified that the chemokine SDF-1α and its receptor CXCR4 play an important role in guiding cell migration in many types of cells including NS/NPCs. The current study tested if SDF-1 could be delivered through alginate to attract and guide migration of NS/NPCs and its progeny both in vitro and in vivo. Using a Boyden chamber migration assay, we found SDF-1α either added directly in the medium or incorporated into alginate threads was capable of influencing migration of cultured NS/NPCs in a dose-dependent manner. In the in vivo study, when injected directly into the cerebral cortex, SDF-1  showed limited capability in inducing neuroblasts migration off the normal tract to the site of SDF-1 injection. When SDF-1 was delivered via alginate thread to the focal injury site at 2 days post TBI, significantly increased number of migrating neuroblasts derived from the SVZ was observed around the injury site. Increased expression of SDF-1 receptor CXCR4 was observed in the NS/NPCs in the SVZ and around the injury site following TBI. These data suggest that bioactive SDF-1α can be delivered via alginate thread and exogenous delivery of SDF-1α and its interaction with receptor CXCR4 mediates migration of newly generated neurons from the SVZ to the site of injury following TBI. Collectively, our study indicates that SDF-1α could be utilized as a guidance cue for tissue repair following brain injury.

L'immunité lactogène protège les muqueueses du nouveau-né, notamment grâce à la richesse en IgA des sécrétions mammaires. Nous avons cherché à purifier par ultrafiltration et RP-HPLC le facteur chémoattractant dans le lait de truie pour les plasmocytes mammaires. Ainsi, nous avons mis en évidence plusieurs facteurs ; parmi ceux-ci, nous avons caractérisé par spectométrie de masse un peptide issu de la protéine SAA (sérum amyloid A) produite par la cellule épithéliale mammaire, et ayant une activité chémoattractante pour les cellules B, suggérant que la SAA participe au recrutement des plasmocytes pendant la lactation et indiquant une nouvelle fonction à cette protéine. La cinétique d'expression des ARNm de la SAA se caractérise par une expression plus importante lors de la lactation que lors de la gestation, suggérant une régulation hormonale. Nos résultats indiquent aussi que VCAM-1, [a]4β1 et MEC pourraient être impliquées dans le recrutement des plasmoblastes à IgA dans la mammelle
Lactogenic immunity protects the mucous membranes of the newborn notably thanks to the wealth in IgA of the mammary secretions. We looked to cleanse by ultrafiltration and RP-HPLC the chemoattractant factor in the milk of sow for the mammary plasma cells. Several chemoattractant factors were found, among which, we characterized by mass spectometry a peptide from SAA protein (serum Amyloid A) producted by mammary epithelial cells, and having a chemoattractant activity for B cells, suggesting that SAA participates in the recruitment of these cells during the lactation, indicating a new function of this protein. The kinetics of expression of the mRNA of SAA were superior in the lactation than during gestation, suggestion a hormonal regulation. Our results also indicate that VCAM-1, [a]4β1 and MEC could be involved in the recruitment of IgA plasma cells in the mammary gland

Introdução: A nefrite lúpica (NL) é o maior preditor de morbidade e mortalidade em pacientes portadores de lupus eritematoso sistêmico. Recentes estudos mostram que a proteína quimiotática de monócitos (MCP-1) está implicada na ativação de células inflamatórias, afetando a progressão e a severidade da NL, e que a excreção urinária do MCP-1 (uMCP-1) está aumentada em pacientes com NL em atividade. Na literatura os dados sobre o polimorfismo do gene MCP-1 A(-2518)G e do seu receptor CCR2 V(-64)I sobre a susceptibilidade para nefrite lúpica ainda estão em discussão. Objetivos: Avaliar a associação entre o polimorfismo do gene MCP-1 e do seu receptor CCR2 em pacientes com NL e indivíduos saudáveis, além da associação de ambos os polimorfismos com parâmetros clínicos e histológicos nos pacientes portadores de NL. Além disso, avaliar a associação entre a excreção urinária do MCP-1 em pacientes portadores de nefrite lúpica em atividade com parâmetros clínicos e histológicos. Pacientes e Métodos: As genotipagens do MCP-1 e do CCR2 foram realizadas em 197 pacientes com nefrite lúpica através da extração do DNA genômico, seguido da técnica de reação em cadeia da polimerase, utilizando-se primers específicos. A dosagem urinária do MCP-1 foi realizada em 34 pacientes com nefrite lúpica em atividade através da técnica de ELISA. Resultados: Foram estudados 197 pacientes portadores de nefrite lúpica, do sexo feminino, com idade média de 28±9,8 anos, sendo 65,5% de etnia branca e 34,5% não-branca, acompanhados em nosso ambulatório durante o período de 69±37,1 meses. Como grupo controle, utilizou-se um grupo de 220 indivíduos saudáveis do sexo feminino, pareados de acordo com idade e etnia. Quanto à distribuição do genótipo do MCP-1, evidenciou-se que a freqüência do genótipo GG foi significativamente maior nos pacientes portadores de nefrite lúpica quando comparado ao grupo controle (12,7%x5,0%) (p=0,019), enquanto que o genótipo AA apresentou maior freqüência no grupo controle, porém sem significância estatística (48,7%x56,8%). Com relação aos alelos, a freqüência do alelo A foi significativamente maior no grupo controle (75,9%x68%) (p=0,007) quando comparada aos pacientes com NL. Já em relação ao polimorfismo do CCR2, não foi observada nenhuma diferença na freqüência do genótipo entre os dois grupos, porém foi observada maior freqüência do alelo V no grupo controle (89,8%x86,3%) (p=0,046). Não houve associação entre o genótipo e alelos do MCP-1 e do CCR2 com a função renal no início e no final do estudo, marcadores imunológicos, manifestações clínicas (SLEDAI) e a classe histológica. Porém, observou-se um predomínio significante dos flares moderado e grave nos pacientes portadores dos genótipos AA e AG (p< 0,05) em relação ao genótipo GG, enquanto que, em relação à distribuição alélica do MCP-1 e ao CCR2, não se notou diferença estatística. Não se evidenciou diferença estatística entre as curvas de sobrevida renal funcional dos pacientes portadores de nefrite lúpica e os genótipos do MCP-1 e CCR2 e seus respectivos alelos. Notou-se diferença estatística na variação da creatinina sérica ao longo do seguimento (p<0,001). Foram também estudados 34 pacientes portadores de nefrite lúpica em atividade, do sexo feminino, com idade média de 28,4 ± 9,9 anos, sendo 26,5% pacientes de etnia branca e 73,5% de etnia não-branca. A dosagem do MCP-1 urinário foi realizada no início do quadro e após 3 e 6 meses de seguimento. Em relação ao uMCP-1, houve um aumento significante do mesmo no início do quadro renal quando comparado com 3 e 6 meses de tratamento (p<0,05). Evidenciou-se um aumento do uMCP-1 nos pacientes que apresentavam creatinina plasmática inicial > 1,2mg/dl (p<0,05), porém não houve associação entre uMCP-1 e a creatinina após 6 meses de tratamento. Não se observou associação entre os níveis de uMCP-1 com as manifestações clínicas (SLEDAI), classe histológica e marcadores imunológicos, exceto quanto ao anticorpo antifosfolípide, pois houve excreção aumentada do uMCP-1 em pacientes com anticorpo antifosfolípide positivo no início do quadro (p<0,05). Notou-se valores elevados do uMCP-1 nos pacientes que apresentaram flares grave e moderado em relação ao flare leve (p<0,05). Quanto à distribuição genotípica do MCP-1 em relação ao uMCP-1, foi observado uma associação do uMCP-1 em pacientes portadores dos genótipos AG e AA quando comparados ao genótipo GG (p<0,05). Já em relação à distribuição genotípica e alélica do CCR2, não se notou nenhuma diferença na freqüência dos mesmos e a dosagem de uMCP-1. Conclusões: Houve uma significante associação do genótipo GG do polimorfismo do MCP-1 em pacientes portadoras de NL na população estudada, além de uma associação entre os níveis do uMCP-1 com a severidade do flare renal e a função renal nas pacientes portadoras de NL.
Introduction: Lupus Nephritis (LN) contributes substantially to morbidity and mortality in patients with systemic lupus erythematosus. Literature data show monocyte chemoattractant protein (MCP-1) is implicated in the activation of inflamatory cells and has been suggested to affect the progression and severity of lupus nephritis and urinary MCP-1 levels (uMCP-1) are increased in LN patients during active renal disease. Literature data about genotype polymorphism of MCP-1 A(-2518)G and of its receptor CCR2 V(-64)I and susceptibility to LN is still open to discussion. Objectives: The aim of our protocol was to study association of the genotype polymorphism of MCP-1 and CCR2 with LN compared to a healthy matched population and study association these polymorphisms with clinical and histological parameters in LN patients. Moreover, investigate the relationship of uMCP-1 on the onset, severity and resolution of LN flare. Patients and Methods: Genomic DNA was extracted from peripheral leukocytes from 197 LN patients and MCP-1 and CCR2 genomic variants were detected by polymerase chain reaction followed by restriction enzyme-fragment analysis. uMCP-1 levels were mesured by enzyme-linked immunosorbent assay from 34 LN flare patients. Results: One hundred and ninety seven (197) female patients with histological diagnosis de LN undergoing follow up in our institution and 220 ethnically matched healthy controls were enrolled in this study. Epidemiological characteristics of the LN group were: age 28±9.8 years, race 65.5% of caucasians and 34.5% of Brazilian afro-south-latins. Baseline values were collected at the onset of LN and final values in their last follow up (69±37.1 months). There was a significant association of the GG genotype polymorphism of MCP-1 with LN patients compared to controls (12.7%x5.0%) (p=0.019), while the allele A distribuition was associated with healthy controls (75.9%x68%) (p=0.007). Considering CCR2 -64 V/I polymorphism genotype there was a association of the allele V with the control group compared to LN (89.8%x86.3%) (p=0.046). Analyzing genotype polymorphism of MCP-1 and CCR2 there werent correlation with renal function, immunological markers, clinical manifestations (SLEDAI) or histological classes of LN. There was a significant association of the AA and AG genotypes polymorphism of MCP-1 with moderate and severe renal flares compared to GG genotype polymorphism of MCP-1 (p< 0.05). Kaplan-Meier analysis of the renal survival curves with respect to the studied genotypes did not show any influence in the progression of renal disease. There was a significant association of the creatinine onset and on follow up (p<0.001). Thrity four (34) female patients with criteria for active LN and histological diagnosis were enrolled and treated for six months. Each patient was evaluated once a month and uMCP-1 bimonthly. Epidemiological characteristics of the group showed: age 28.4±9.9 years and race 26.5% caucasians and 73.5% Brazilian afro-south-latins. uMCP-1 excretion at onset (T0) of LN was significantly increased when compared to uMCP-1 measured on the third (T3) and sixth months (T6) (p<0.05). Analyzing uMCP-1 values on T0 there was a correlation with creatinine (p<0,05), but not with, clinical manifestations histological classes of LN or immunological markers, except in patients with positive antiphospholipid autoantibodies demonstrated increased of uMCP-1 (p<0.05). Otherwise, uMCP-1 levels were associated with seriousness of nephritis flares, severe and moderate over mild (p<0.05). Considering MCP-1 polymorphism genotype there was association of the AA and AG genotypes with increased uMCP-1 in patients with active renal disease (p<0.05). Conclusions: There is a significant association of the GG genotype of MCP-1 -2518 A/G polymorphism with LN in our population. uMCP-1 levels in LN is associated with flare seriousness and renal function.

Background: There is need for judicious use of immunosuppression in patients with active lupus nephritis (LN), however this is guided by renal biopsy which is invasive and not freely available in most centres. Novel urinary biomarkers such as monocyte chemoattractant protein-1 (MCP-1) and tumour necrosis factor-like weak inducer of apoptosis (TWEAK) are secreted in the kidney and may be useful for predicting histological class, monitoring flares and assessing response to therapy. We assessed the utility of urinary MCP-1 (uMCP-1) and TWEAK (uTWEAK) in predicting renal histological findings, disease flares and treatment response 6 months following initiation of treatment for LN in newly biopsied patients. Methods: We recruited consenting patients with active LN confirmed on kidney biopsy. Relevant baseline demographic, biochemical and histological information was collected from the patients. ELISA methods were used to assess uMCP-1 and uTWEAK at baseline and at 6 months after completion of induction therapy. Results: There were 14 females and 6 male patients with a mean age of 29.8 ± 10.7 years, 60% were of mixed ancestry, 70% had proliferative LN. There was no association between uMCP-1 and uTWEAK and histological features (LN class, activity index, chronicity index and interstitial fibrosis). At 6 months, 6 patients were lost to follow-up and of the remaining 14, 12 (85%) attained remission (partial remission (n = 7) or complete remission (n = 5)). Both biomarkers were elevated in patients with active disease and significantly declined amongst those attaining remission, p = 0.018 and p = 0.015 respectively. However, for those not attaining remission, no association was found for both biomarkers (p >0.05). Conclusion: Our study did not show correlation between uMCP-1 and uTWEAK with histological features of LN. However, both biomarkers were elevated in patients with active disease and correlated with the remission status at the end of induction phase of treatment.