Immunological mediators are thought to be responsible for many of the pathophysiologic changes observed in human glomerulonephritis. Previous studies have shown the importance of immunological mediators such as immune-complexes, complement and neutrophils. Recently, a significant role for the monocyte/macrophage system in glomerular injury has been emphasized by several investigators. The present study was designed to investigate the effect of macrophage supernatant factor(s) on glomerular cell metabolism in an in vitro tissue culture system. Human glomerular cells are grown in vitro and were characterized by their phagocytic capacity and their morphologic characteristics. Light microscopy, scanning and transmission electron microscopy, and observation of their growth pattern revealed two basic cell types: an epithelial and a mesangial cell. Epithelial cells were represented by large (100-200 μ in length) and small (50-70 μ in length) flat polygonal cells. The larger epithelial cell was primarily seen in the initial outgrowth and was not easily maintained in culture. Therefore they were not used for the metabolic experiments. On the other hand, the smaller epithelial cell was maintained in culture for an average of 5 to 8 passages. The mesangial cells were medium-sized (75-120 μ in length), of variable morphology but mostly spindle-shaped and grew in a characteristic storiform pattern. Both cell types kept their morphologic appearance with subculturing and cryopreservation. Cultured glomerular cells were treated with dialyzed macrophage supernatants obtained from mouse or human peripheral blood monocytes. Undiluted or diluted macrophage supernatants were over-layed on glomerular cells cultured in 96-well flat-bottom microtiter plates. DNA, RNA and protein synthesis were evaluated by incorporation of radio-labelled precursors. Macrophage supernatants failed to stimulate DNA synthesis in epithelial cells as measured by incorporation of 3HTdR. The same macrophage supernatant did, however, significantly increase the uptake of 3HUdR and a 14-C amino acid mixture, indicating an increase in RNA and protein synthesis. The results with DNA metabolism are consistent with in vivo observations in that epithelial cells are not regarded as the intrinsic proliferating cell in the hypercellularity observed in glomerular injury. The stimulation of RNA and protein synthesis may be related to the in vivo thickening of the glomerular basement membrane. In addition, it may be related to the production of molecules which may directly or indirectly affect endothelial or mesangial cells and/or affect the local charges in the glomeruli which are known to be important in permeselectivity of the capillary wall. In the case of mesangial cells, exposure to macrophage supernatants led to a significant increase in DNA snythesis as measured by the increase in uptake of 3HTdR. No stimulation was seen in RNA and protein synthesis as measured by the radioactive label technique. The increase in DNA synthesis correlates with in vivo observations of mesangial cell proliferation in glomerular injury. The factor(s) in the macrophage supernatant which affect the metabolism of glomerular cells in vitro is non-dialyzable and denatured by freezing and thawing. In addition, preliminary results indicate that the activity stimulating RNA and protein synthesis (epithelial cells) is insensitive to heat treatment while the one affecting DNA synthesis (mesangial cells) seems to be sensitive to heat treatment. Neither was shown to be species specific since both human and mouse macrophage supernatant induced the same changes in glomerular cell metabolism. The results of this investigation suggest that both cell types are selectively affected by a factor(s) present in the macrophage supernatant. It is likely that more than one factor is responsible for the metabolic changes observed. The possibility that this factor(s) may be identical with macrophage factors previously described in other cell systems cannot be ruled out at this time. These in vitro observations further support an active role for the monocyte/macrophage system in glomerular injury in experimental and clinical glomerulonephritis.

Thesis (M.Sc.)--Victoria University of Wellington, 2009. / "Malaghan Institute of Medical Research." Includes bibliographical references.

Thesis (Ph.D.)--Cleveland State University, 2009. / Abstract. Title from PDF t.p. (viewed on Sept. 28, 2009). Includes bibliographical references (p. 84-90). Available online via the OhioLINK ETD Center and also available in print.

No description available.

La maladie d'Alzheimer (MA) et l'angiopathie amyloïde cérébrale (AAC) sont les deux formes les plus courantes de démence liée à l'âge qui partagent de nombreuses caractéristiques moléculaires, notamment l'accumulation de bêta-amyloïde (Aβ) dans les parois des vaisseaux sanguins cérébraux. Les cellules du système immunitaire inné, telles que les monocytes patrouilleurs, sont capables de surveiller les vaisseaux sanguins cérébraux et de phagocyter la bêta-amyloïde vasculaire ainsi que d'autres substances. Les monocytes patrouilleurs sont devenus une cible thérapeutique dans la MA, et leur phagocytose de l'Aβ permettrait une redistribution à l'équilibre entre le parenchyme et les espaces périvasculaires et vasculaires, ce qui réduirait ensuite la charge dans le parenchyme. Des recherches antérieures ont démontré que les monocytes peuvent être convertis du phénotype inflammatoire au phénotype de patrouille en utilisant la liaison du MDP à NOD2 (Lessard et al., 2017). Nous émettons l'hypothèse que le développement d'analogues de la MDP ayant des effets immunomodulateurs similaires à ceux de la MDP pourrait conduire à un médicament préventif dans la MA. Nous avons utilisé les lignées cellulaires HEK-Blue NOD2 et HEK-Blue TLR2 pour détecter les analogues qui se lient à NOD2. Nous avons également utilisé le test MTS sur des PBMC et des cellules HepG2 pour évaluer la viabilité cellulaire et la cytométrie perlée pour caractériser les cytokines et les interférons libérés par les cellules exposées aux analogues du MDP. Nous avons effectué des tests de phagocytose pour évaluer si les analogues du MDP modifiaient le taux de phagocytose par les monocytes. De plus, nous avons effectué des tests in vivo sur des souris WT pour évaluer si les analogues de la MDP pouvaient provoquer des changements phénotypiques dans les monocytes et si ces changements phénotypiques se produisaient chez les souris NOD2 KO. Les analogues de la MDP ont le potentiel de devenir un médicament préventif de la MA en augmentant la phagocytose de l'Aβ et en diminuant l'Aβ vasculaire. D'autres recherches sont nécessaires pour mieux comprendre le rôle exact des monocytes patrouillant dans la MA.

Contexte. Les traitements immunosuppresseurs actuels sont associés à un risque considérablement accru d'infection grave et de cancer. Un outil identifiant les patients surimmunosupprimés (SI) pourrait changer de façon significative le suivi clinique et réduire les comorbidités liées à l’immunosuppression. Méthodes. Dans cette étude observationnelle prospective, plus de 300 collections de cellules mononucléées du sang périphérique (PBMCs) ont été prélevées longitudinalement au cours des 2 premières années post-transplantation chez 50 greffés rénaux. Les patients ont été classés comme SI ou contrôles. 2 patients ont été exclus à cause d’une perte de greffe, d’un risque biologique et 7 autres dus à un problème de congélation des cellules en début d’étude. Les PBMC ont été incubées pendant une nuit (non stimulées, stimulées au LPS ou des peptides de l'EBV). La réponse monocytaires a été examinée par cytométrie en flux. Résultats. Les patients ont été répartis au hasard dans une cohorte d’entrainement (n=12) et une cohorte de validation (n=29). Dans la cohorte d’entrainement, nous avons exploré l'expression de surface et de la production intracellulaire de cytokines pro-inflammatoires dans les sous-populations monocytaires. Dans les cultures stimulées par les peptides de l’EBV, les monocytes de patients SI ont présenté une réponse TNFa dans la sous-population CD14+CD16+ (pro-inflammatoire) inférieure aux patients contrôles. Un seuil de 73% des monocytes CD14+CD16+ positifs au TNFa+ a été déterminé par courbe ROC. Une règle de classification a été définie: tout patient avec 2 points consécutifs sous le seuil du %TNFa pour les monocytes CD14+CD16+ a été classé SI. Dans la cohorte d’entrainement, cette règle a donné une sensibilité=83%, spécificité=83%, VPP=83% et VPN=83%. Dans la cohorte de validation, les valeurs ont été sensibilité=90%, spécificité=63%, VPP=56% et VPN=92%. Conclusion. Cette étude démontre que la mesure de la réponse des monocytes après stimulation in vitro des PBMCs peut informer sur l'état de surimmunosuppression. / Background. The current immunosuppressive regimens are associated with a substantially increased risk of serious infection and cancer. A tool that identifies over-immunosuppressed (OIS) patients could significantly change clinical management and reduce comorbidities related to the immunosuppression. Methods. In this prospective, observational study, over 300 peripheral blood mononuclear cells (PBMC) samples were collected longitudinally during the first 2 years post-transplant in 50 kidney recipients. Patients were classified as OIS [opportunistic infection (n=16), BK viremia or nephropathy (n=12), CMV viremia (n=1), ≥3 recurrent bacterial infection (n=2), cancer (n=1)] or controls (n=25). 2 patients were excluded because of graft loss, biohazard issue or 7 because of technical issue with the cells frozen in the first months of the protocol. Whole PBMCs were incubated overnight (unstimulated, LPS-stimulated and EBV peptides-stimulated) and the monocyte response was examined by flow cytometry. Results. Patients were randomly distributed in a training (n=12; 6 OIS and 6 controls by design) and a validation cohort (n=29; 10 OIS and 19 controls). In the training cohort, we explored the surface expression of intracellular production of pro-inflammatory cytokines in monocyte subsets. In the EBV peptides-stimulated cultures, monocytes from OIS patients had a lower TNFα response in the CD14+CD16+ (pro-inflammatory) subset. Cut-off of 73% CD14+CD16+ monocytes positives for TNFα was determined by ROC curve. A classification rule was defined: any subject with 2 consecutive samples under the %TNFα+ cutoff for CD14+CD16+ monocytes was considered OIS. In the training cohort, this rule yielded the following: sensitivity=83%, specificity=83%, positive predictive value=83% and a negative predictive value=83%. In the validation cohort, values were: sensitivity=90%, specificity=63%, positive predictive value=56% and negative predictive value=92%. Conclusion. This proof-of-concept study demonstrates that measuring monocyte response when whole PBMCs are stimulated in vitro can inform on the OIS status.

PACAP et VIP sont deux peptides structuralement reliés qui appartiennent à la classe de neuropeptides comprenant la sécrétine, le glucagon et le growth hormone releasing factor. VIP et PACAP ont de nombreuses fonctions biologiques au niveau du système nerveux et sont considérés comme des agents neurotransmetteurs, sécrétagogues, neuroprotecteurs, neurotrophiques, mitogéniques et comme agents différenciant. <p><p>VIP et PACAP utilisés à des concentrations de l’ordre du nano-molaire sont décrits comme agents anti-inflammatoires. Dans cette thèse, nous montrons que les neuropeptides PACAP et VIP utilisés à des doses élevées de l’ordre du μ-molaire agissent également comme des molécules pro-inflammatoires au niveau des monocytes humains.<p><p>Nous montrons aussi qu’au sein des monocytes humains, le PACAP agit par l’intermédiaire du récepteur VPAC-1. Les voies de signalisation activées incluent de façon proximale la PLC et la PI3-kinase et de façon plus distale les voies ERK et p38 des MAP-Kinases et les voies dépendantes de focal adhésion kinase associées à la protéine du cytosquelette paxilline. PACAP induit également un pic calcique résultant d’une mobilisation intra- et extracellulaire de calcium.<p><p>Au niveau fonctionnel, nous montrons quelle PACAP augmente la production de radicaux libres et l’expression membranaire de l’intégrine CD11b impliquée dans l’adhésion et dans la régulation de nombreuses voies métaboliques.<p><p>Nous montrons également que de faibles doses de PACAP de l’ordre du nM sont suffisantes pour désensibiliser les monocytes à des concentrations plus importantes de PACAP, concentrations connues pour avoir un effet pro-inflammatoire. Le PACAP a ainsi un double rôle, anti-inflammatoire à faible dose et pro-inflammatoire à dose plus élevé.<p><p>Nous avons de plus investigué la présence d’une transactivation au niveau des cellules monocytaires. Dans ces cellules, PACAP comme rapporté dans les cellules neuronales, utilise la signalisation NGF/TrkA. PACAP augmente la fraction phosphorée du récepteur TrkA. L’utilisation d’inhibiteur spécifique du récepteur au NGF est associée à une diminution de la phosphorylation des voies PACAP-dépendantes telles que AKT et ERK. L’inhibiteur du récepteur au NGF diminue également la mobilisation calcique induite par le PACAP avec au niveau fonctionnel une diminution de la production de radicaux libres et de l’expression de l’intégrine CD11b. Enfin, l’observation que les voies métaboliques (ERK) et que les fonctions (production de radicaux libres) induites par le NGF au sein des cellules monocytaires est sensible à la pertussis toxine, agent modulant des récepteurs à protéines G, indique que les phénomènes de transactivation entre les voies PACAP/VPAC-1 et NGF/TrkA se font de façon bidirectionnelle.<p><p>Nous montrons enfin que ;à la fois PACAP et VIP sont des ligands du récepteur proinflammatoire FPRL1 au sein des monocytes induisant l’activation des voies AKT/ERK et la mobilisation calcique, responsables de l’augmentation d’expression de l’intégrine CD11b.Nous montrons cependant qu’il existe une voie propre au récepteur VPAC-1, indépendante de FPRL1, médiée par l’axe cAMP/PKA/p38 responsable spécifiquement d’une activité proinflammatoire avec sécrétion de MMP9 et augmentation membranaire de CD35. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Acute myocardial infarction (AMI) results in the activation of the innate immune system with monocytes playing critical roles in both the initial inflammation following myocardial ischaemia and subsequent recovery. Monocytes are a heterogeneous cell population and observations from experimental models demonstrate that immediately following myocardial injury, classical inflammatory monocytes, which are highly phagocytic, are recruited to ischaemic myocardium from the bone marrow and spleen and peak at 48 hours. This is followed by the recruitment of non-classical monocytes that are involved in repair and healing, peaking at day 5. The monocyte response in humans following AMI is currently poorly understood. Due to their central role in the pathogenesis of AMI, monocytes are attractive both as potential biomarkers to inform of extent of myocardial injury (and recovery) and also as therapeutic targets with the specific targeting of monocytes in experimental models resulting in reduced infarction size and improved LV remodelling. However, in spite of these promising results and our greater understanding of the pathogenesis of AMI, no immune-modulating therapeutic has been translated into routine clinical practice. We therefore hypothesized that characterisation of the monocyte response to AMI by flow cytometry and gene expression profiling in both experimental models and humans would give novel insights into underlying biological processes and function (both locally in the myocardium and systemically), identify novel therapeutic targets, enable their use as cellular biomarkers of disease, and test conservation between species validating the experimental model for future investigation. Classical inflammatory monocytes were found to significantly increase in the peripheral blood 48 hours following AMI in both mice and humans, with the magnitude of the monocyte response correlating with the extent of myocardial injury in both species. Gene expression profiling of peripheral circulating monocytes following AMI identified a number of candidate genes, biological pathways and upstream regulators that were conserved between species and that could represent novel therapeutic targets. Furthermore, in an experimental model of AMI, leukocytes appeared to have effects beyond the ischaemic myocardium, with leukocyte recruitment in remote myocardium and in kidneys associated with elevated inflammatory markers and endothelial activation.

Background: Acute lung injury (ALI) and nosocomial pneumonia are major causes of morbidity and mortality. There are 200,000 cases per year of ALI in the US with a mortality of 40%. On the intensive care unit (ICU), ALI accounts for over 40% of all ventilated patients at any one time. Despite this huge burden on healthcare and the relatively high prevalence, no therapies currently exist in clinical practice that attenuate the condition. The pathophysiology and aetiology of ALI is multifactorial but neutrophilic influx and consequent damage to the endothelial-epithelial interface are regarded as central features. Alongside neutrophils, peripheral blood monocytes (PBMs) are recruited to the acutely inflamed lung. The role played by PBMs in perpetuating the pathogenic neutrophilic influx remains poorly characterised. Nosocomial pneumonia is also a major problem with drug resistant organisms. With the increasing prevalence of antibiotic resistance and the paucity of novel antimicrobials being generated by pharmaceutical companies, there is real concern that the end of the ‘antibiotic era’ may be approaching. AIMS 1) To develop murine models of lung inflammation and infection 2) To establish the role of the PBM in perpetuating the neutrophilic response in ALI 3) To develop non-invasive methodologies to study the trafficking of cells and molecular events within the inflamed lung 4) To apply a novel antimicrobial to prevent and treat nosocomial pneumonia Methods: A murine model of ALI was utilised using direct intratracheal instillation of lipopolysaccharide. To this model 3 different PBM depletion strategies were applied to study the effect on neutrophil recruitment and consequent lung injury. Non invasive optical imaging was utilised to study the effect of PBM depletion on proteolytic events within the murine lung. To understand cellular trafficking, cell labeling strategies were compared for primary murine macrophages with whole body optical imaging in mice. Murine models of Staphylococcus aureus, Pseudomonas aeruginosa and Burkholderia cepacia were established and a novel antimicrobial agent called the nonalysine like peptoid (NLLP) tested in vitro and in vivo for efficacy. Results: PBM depletion significantly attenuated neutrophil recruitment in an established model of ALI. Near infrared (NIR) optical imaging permitted the non invasive tracking of primary murine cells. A non toxic peptidomimetic agent (NLLP) possessed antimicrobial activity against gram positive and gram negative pathogens with therapeutic and prophylactic efficacy in vivo. Conclusions: PBM depletion is a potential therapeutic strategy for treating ALI. Further studies are required to determine the exact mechanism by which PBMs orchestrate neutrophil recruitment. Optical imaging is a versatile platform for molecular imaging. A novel antimicrobial agent termed NLLP has been discovered with therapeutic and prophylactic efficacy against multi-drug resistant pathogens.

published_or_final_version / Pharmacology / Master / Master of Philosophy