The role of inflammatory chemokine receptors in hypertension and blood pressure regulation

Chemokines regulate the migration of leukocytes through binding to chemokine receptors. The inflammatory CC chemokine receptors CCR1, CCR2, CCR3 and CCR5 (iCCRs) are expressed by several different leukocytes and are important in orchestrating inflammatory responses. These receptors can bind to multiple CC chemokines and many of these chemokines share receptors, resulting in a highly complex system in which the exact role of each receptor is not understood. To aid understanding of these receptors, two unique mouse strains have been developed. These are mice that are deficient for all four iCCRs (iCCR-KO) and mice that express fluorescent reporter proteins for each of the iCCRs (REP mice). Hypertension is when blood pressure (BP) is elevated above 140/90 mmHg. There is evidence for a role of inflammation in the development of hypertension and the consequent end organ damage which increases the risk of cardiovascular disease. Several chemokines and chemokine receptors have been implicated in hypertension but due to the complexity of the chemokine system, the role they play in the pathogenesis of the disease is unclear. Therefore the role of iCCRs in hypertension was investigated using iCCR-KO and REP mice. WT, REP and iCCR-KO mice were subject to 7 or 14 days of Angiotensin (Ang) II induced hypertension. iCCR expression was characterised in REP and WT mice and the effect of iCCR deficiency on BP, vascular function, inflammation and cardiac and vascular remodelling was assessed using the iCCR-KO mice. Aortic CCR2 and CCR5 expression increased in Ang II treated WT mice compared to control WT mice. Further, iCCR-KO mice were protected from Ang II induced vascular dysfunction but iCCR deficiency did not influence BP or remodelling. iCCR-KO mice were also shown to have altered circulating leukocyte populations in Ang II induced hypertension. Control iCCR-KO mice tended to have a lower BP than WT mice so the effect of iCCR deficiency on regulators of BP in the kidneys and kidney leukocyte infiltration was investigated. iCCR-KO mice had fewer inflammatory monocytes and reduced mineralocorticoid receptor mRNA expression. This could influence BP but further studies are needed. Overall, novel mouse models have been used to identify how iCCRs are involved in hypertension. The results described here suggest that iCCRs, in particular CCR2 and CCR5, are involved in regulating vascular dysfunction in hypertension. Through improving understanding of these receptors in the disease, there is increased potential to target them as treatments that would ultimately reduce the risk of cardiovascular disease

The novel role of epidermal growth factor (EGF) in the regulation of ion channels in the calu-3 submucosal cell line

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cell membrane bound chloride ion channel regulated by cyclic AMP-dependent phosphorylation and levels of intracellular ATP. Mutations in this channel, such as the common deletion of phenylalanine at residue 508 (CFTRΔF508), leads to a decrease in chloride transport seen in the disease condition cystic fibrosis (CF). The mutant CFTR is not processed in the normal way and consequently not delivered to the cell membrane. Currently, the effect of growth factors such as epidermal growth factor (EGF) on ion transport in the airway has not been previously researched and is consequently unknown. Therefore the aim of this thesis is to determine (i) if EGF has an effect on ion transport in the submucosal cell line Calu-3, (ii) what the mechanisms are behind this, and (iii) if the effect of EGF was due to induction of gelatinase activity or a transactivation process. Functional investigations looking at ion transport were carried out by using short circuit current. This technique was complemented by traditional molecular biology techniques such as RT-PCR, Western blotting, flow cytometry and gelatin zymography. The level of EGF, a potent inducer of gelatinases, is known to be elevated in the lungs during tissue repair in CF. Calu-3 cells preincubated with EGF on the basolateral membrane increased initial current at one hour via a EGFR-PI3K-PKC-δ-KCNN4/KCNQ1 signalling pathway. Similarly, preincubation with EGF also decreased forskolin induced short circuit current compared to untreated monolayers at 1 to 3 hours, with a recovery at 24 hours. The decreases were found to be dependent on the activation of KCNQ1 since chromanol 293B, a specific inhibitor for KCNQ1, restored the short circuit current back to untreated levels. Stimulation of the β2 adrenergic receptors with salbutamol were not reduced using metalloproteinase inhibitor, GM-6001 and EGFR inhibitor, AG1478. This suggested that stimulation of β2 adrenergic receptors does not lead to transactivation of EGFR via activation of sheddases and the release of EGF ligand. β3 adrenergic receptors are present in Calu-3, but produce negligible currents when stimulated. It was concluded that EGF induced potassium channel activation led to a change in chloride driving force. This activation of potassium channels has previously been linked to wound repair in the airway during disease. The implications of this study suggest that manipulation of the EGF signalling pathway and / or potassium channel activity in the lungs may be beneficial in disease conditions such as CF for increasing chloride transport

Investigating the mechanism of renal cystogenesis in tuberous sclerosis and polycystic kidney disease

Tuberous sclerosis (TSC) is an autosomal dominant disorder caused by germline mutations in either TSC1 or TSC2 and characterised by the development of benign hamartomatous growths in multiple organs and tissues. Clinical trials are underway for the treatment of TSC-associated tumours using mammalian target of rapamycin (mTOR) inhibitors. Here, we show that many of the earliest renal lesions from Tsc1+/ and Tsc2+/ mice do not exhibit mTOR activation, suggesting that pharmacological targeting of an alternative pathway may be necessary to prevent tumour formation. Patients with TSC often develop renal cysts and those with inherited co- deletions of the autosomal dominant polycystic kidney disease (ADPKD) 1 gene (PKD1) develop severe, early onset, polycystic kidneys. Using mouse models, we crossed Tsc1+, and Tsc2+I mice with Pkd1+/ mice to generate double heterozygotes. We found that Tsc1+lPkd1+, and Tsc2+l Pkd1+, mice had significantly more renal lesions than their corresponding single heterozygote littermates indicating a genetic interaction between Tsd and Tsc2 with Pkd1. In agreement with our findings from Tsc1+/ and Tsc2+/ mice, we found that a large proportion of cysts from Tsc1+l Pkd1+, and Tsc2+l Pkd1+, mice failed to stain for pS6, suggesting that initiation of renal cystogenesis in these animals may occur independently of mTOR activation. We analysed primary cilia in phenotypically normal renal tubule epithelial cells by scanning electron microscopy (SEM) and found that those from Tsc1+, and Tsc2+I mice were significantly shorter than those from wild-type littermates (2.122pm and 2.016pm vs. 2.233pm, respectively, P<0.001). Primary cilia from epithelial cells lining renal cysts of Tsc1+' and Tsc2+I' mice were consistently longer (5.157pm and 5.091pm respectively). Interestingly, we found that Pkd1- deficiency coupled with either Tsd or 7sc2-deficiency altered the length of the primary cilia from both normal renal tubule cells (restored to 'wild-type' length) and epithelial cells lining cysts (Tsc1+tPkd1+, Mean 3.38pm and Tsc2+,Pkd1+l Mean 3.09pm). These novel data demonstrate that the Tsc and Pkd1 gene products help regulate primary cilia length which may prevent renal cystogenesis. Consistent with the observation that primary cilia modulate the planar cell polarity (POP) pathway, we found that many dividing pre-cystic renal tubule epithelial cells from Tsc1+/ , Tsc2+/ and Pkd1+/ mice were highly misorientated along the tubule axis. This could potentially lead to tubule dilation and subsequent cyst formation. We therefore propose that defects in cell polarity underlie both TSC and ADPKD-associated renal cystic disease and targeting of this pathway may be of key therapeutic benefit.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Investigating the mechanism of renal cystogenesis in tuberous sclerosis and polycystic kidney disease

Tuberous sclerosis (TSC) is an autosomal dominant disorder caused by germline mutations in either TSC1 or TSC2 and characterised by the development of benign hamartomatous growths in multiple organs and tissues. Clinical trials are underway for the treatment of TSC-associated tumours using mammalian target of rapamycin (mTOR) inhibitors. Here, we show that many of the earliest renal lesions from Tsc1+/ and Tsc2+/ mice do not exhibit mTOR activation, suggesting that pharmacological targeting of an alternative pathway may be necessary to prevent tumour formation. Patients with TSC often develop renal cysts and those with inherited co- deletions of the autosomal dominant polycystic kidney disease (ADPKD) 1 gene (PKD1) develop severe, early onset, polycystic kidneys. Using mouse models, we crossed Tsc1+, and Tsc2+I mice with Pkd1+/ mice to generate double heterozygotes. We found that Tsc1+lPkd1+, and Tsc2+l Pkd1+, mice had significantly more renal lesions than their corresponding single heterozygote littermates indicating a genetic interaction between Tsd and Tsc2 with Pkd1. In agreement with our findings from Tsc1+/ and Tsc2+/ mice, we found that a large proportion of cysts from Tsc1+l Pkd1+, and Tsc2+l Pkd1+, mice failed to stain for pS6, suggesting that initiation of renal cystogenesis in these animals may occur independently of mTOR activation. We analysed primary cilia in phenotypically normal renal tubule epithelial cells by scanning electron microscopy (SEM) and found that those from Tsc1+, and Tsc2+I mice were significantly shorter than those from wild-type littermates (2.122pm and 2.016pm vs. 2.233pm, respectively, P<0.001). Primary cilia from epithelial cells lining renal cysts of Tsc1+' and Tsc2+I' mice were consistently longer (5.157pm and 5.091pm respectively). Interestingly, we found that Pkd1- deficiency coupled with either Tsd or 7sc2-deficiency altered the length of the primary cilia from both normal renal tubule cells (restored to 'wild-type' length) and epithelial cells lining cysts (Tsc1+tPkd1+, Mean 3.38pm and Tsc2+,Pkd1+l Mean 3.09pm). These novel data demonstrate that the Tsc and Pkd1 gene products help regulate primary cilia length which may prevent renal cystogenesis. Consistent with the observation that primary cilia modulate the planar cell polarity (POP) pathway, we found that many dividing pre-cystic renal tubule epithelial cells from Tsc1+/ , Tsc2+/ and Pkd1+/ mice were highly misorientated along the tubule axis. This could potentially lead to tubule dilation and subsequent cyst formation. We therefore propose that defects in cell polarity underlie both TSC and ADPKD-associated renal cystic disease and targeting of this pathway may be of key therapeutic benefit

The Effect of Malaysia General Election on Financial Network: An Evidence from Shariah-Compliant Stocks on Bursa Malaysia

Instead of focusing the volatility of the market, the market participants should consider on how the general election affects the correlation between the stocks during 14th general election Malaysia. The 14th general election of Malaysia was held on 9th May 2018. This event has a great impact towards the stocks listed on Bursa Malaysia. Thus, this study investigates the effect of 14th general election Malaysia towards the correlation between stock in Bursa Malaysia specifically the shariah-compliant stock. In addition, this paper examines the changes in terms of network topology for the duration, sixth months before and after the general election. The minimum spanning tree was used to visualize the correlation between the stocks. Also, the centrality measure, namely degree, closeness and betweenness were computed to identify if any changes of stocks that plays a crucial role in the network for the duration of before and after 14th general election Malaysia

The role of endothelial Dll4 in cancer metastasis

Tese de Doutoramento em Ciências Veterinárias, especialidade de Ciências biológicas e biomédicasThe metastatic spread of cancer is still the major barrier to the treatment of this disease. Cancer-mortality is mainly due to recurrence and metastasis. Although much has been done in the field of cancer treatment, prevention and approach to metastases are still areas not fully explored. Over and under-expression of Dll4/Notch signaling has been demonstrated to impair tumor growth through opposing patterns of vascular modulation in different mouse tumor models and human cancer xenografts. Recent evidence implicates Dll4/Notch pathway in the metastasis mechanism, but less is known about the specific role of endothelial Dll4. For this reason, we proposed to investigate how endothelial Dll4 expression interferes with the metastatic process. To address it we used a spontaneous metastasis mouse model based on Lewis Lung Carcinoma (LLC) subcutaneous transplants in endothelial-specific Dll4 lossof- function (eDll4cKO) and endothelial-specific Dll4 over-expression (D4OE) mice. Results demonstrated that eDll4cKO is responsible for the vascular function regression that leads to tumor growth reduction. Early steps of epithelial-to-mesenchymal transition (EMT) and cancer stem cell selection were also inhibited by eDll4cKO, leading to a substantial reduction of circulating tumor cells and reduction in the number and burden of macrometastases. Intravasation and extravasation were also compromised by eDll4cKO, possibly due to blockade of the metastatic niche. In the case of the D4OE mice we observed that tumor growth reduction was achieved by vessel proliferation restriction along with an improved vascular maturation, which allowed a more efficient delivery of chemotherapy. This last effect of vessel normalization seemed to prevent metastasis formation even though EMT markers were increased. In conclusion, despite the opposite vascular architecture phenotypes of eDll4cKO and D4OE, both lead to a reduction in metastasis. This is in line with the concept of Dll4 dosage observed in the wound-healing context and represents a promising therapeutic approach in metastasis prevention/ treatment.RESUMO - A importância da expressão endotelial do ligando Dll4 no processo de metastização - O cancro é, atualmente, uma das principais causas de morte em todo o mundo, a par das doenças cardiovasculares e da doença pulmonar obstrutiva crónica. A mortalidade por cancro está frequentemente associada à metastização, quer seja pelo compromisso hemodinâmico, quer seja pelas consequências do seu tratamento. Apesar dos avanços no tratamento do cancro, com novos agentes biológicos e imunomodeladores, a investigação do mecanismo da metastização, prevenção e abordagem das metástases continuam a ser áreas pouco exploradas. A via Notch é uma via de sinalização intercelular altamente conservada que está envolvida na determinação do destino, regulação da proliferação e diferenciação celulares. O ligando Dll4 tem uma expressão predominantemente endotelial arterial e capilar, crítica para o correto desenvolvimento embrionário, mas restringindo-se às pequenas artérias e capilares no adulto em homeostasia. Ele atua como agente anti-angiogénico em situações de “stress” fisiológico e em contexto tumoral, promovendo a ocorrência de pausas ritmadas na fase de crescimento vascular ativo, que são conducentes à maturação funcional da vasculatura nascente. Os efeitos do aumento e diminuição da expressão do ligando Dll4 sobre a dinâmica tumoral já foram extensamente estudados em modelos tumorais de ratinhos transgénicos e com xenografos. Geralmente, a ativação aberrante da via de sinalização Dll4/Notch está associada a mau prognóstico e probabilidade de metastização. Por outro lado, as terapias de bloqueio Dll4/Notch têm-se revelado eficazes no tratamento de modelos tumorais resistentes às terapias baseadas no “vascular endotelial growth factor” (Vegf), em modelos pré-clínicos. Estudos recentes implicam a ativação da via Notch nas células endoteliais, com alteração do seu fenótipo de forma a favorecer a transmigração das células tumorais, bem como a secreção de “vascular cell adhesion molecule-1”, que promove a adesão de leucócitos e potencia os fenómenos de intravasação e extravasação. Nesse sentido, surge a oportunidade de explorar o papel do ligando Dll4, em concreto, de que forma é que a sua expressão endotelial influencia as várias etapas da cascata da metastização. Para isso, usamos um modelo murino de metastização espontânea que consistiu na injeção subcutânea de células Lewis Lung Carcinoma (LLC) em dois tipos de ratinhos transgénicos: ratinhos com perda-de-função ou sub-expressão endotelialespecífica de Dll4 e ratinhos com ganho-de-função ou sobre-expressão endotelialespecífica de Dll4. Relativamente ao estudo da influência do ganho-de-função endotelialespecífico de Dll4 sobre o tumor primário, foram também usados outros ratinhos: o modelo de papiloma da pele induzido quimicamente e o modelo murino transgénico RIP-Tag2, com insulinoma. Para avaliar a angiogénese do tumor primário foram determinados parâmetros como: densidade, maturidade, funcionalidade e permeabilidade vasculares. Depois de explorado o efeito de Dll4 sobre o fenómeno de transição-epitélio-mesenquimal (EMT), in vitro, procurou-se explorar o mesmo, in vivo, com recurso aos ratinhos mutantes endoteliais-específicos citados. EMT foi caracterizada através da marcação imunofluorescente para Snail-1 e Twist. As células tumorais estaminais (CSC) foram exploradas através da marcação imunofluorescente para p63 e CD49f. A hipóxia tumoral foi avaliada através do teste com “Hypoxyprobe”. O circuito das células tumorais metastáticas foi analisado com recurso à marcação das células com uma proteína verde fluorescente, recorrendo às técnicas de imunofluorescência e “fluorescent associated cell sorting”. A análise da expressão génica foi feita através do “quantitative real time polymerase chain reaction”, tanto nos ratinhos com perda, como ganho-de-função endotelial-específico de Dll4. Quanto ao estudo da perda-de-função endotelial-específica de Dll4 foi possível avaliar, genericamente, todas as etapas da metastização. Começando pelo tumor primário, verifica-se que ocorre uma redução considerável do seu crescimento, em parte devido à angiogénese que, apesar de aumentada, é desorganizada e menos funcional. A árvore vascular apresenta inúmeras ramificações e um revestimento peri-vascular insuficiente (redução de células de músculo liso, dos marcadores α-SMA e Pdgfr-β), indicativo de imaturidade vascular. Observa-se um aumento da permeabilidade, que leva a fenómenos de exsudação e consequente má perfusão do tumor. Este endotélio disfuncional acaba por sofrer regressão, condicionando o crescimento tumoral. Constata-se que eventos iniciais como a EMT e a seleção de clones de células tumorais estaminais (CSC) são inibidos pelo bloqueio endotelial-específico de Dll4, conduzindo a uma redução do número de células tumorais circulantes e do número e crescimento das macro-metástases. Os fenómenos posteriores de intravasação e extravasação estão também reduzidos, provavelmente pela não sinalização do tumor primário à medula óssea, via Dll4/ Notch1/ Hey1/ TGF-β. Pelo que, há uma redução da mobilização de células mieloides Cd11c+/VEGFR-1+ para o nicho metastático (pulmão) e fraca deposição de fibronectina, que seria essencial para a acomodação das células tumorais circulantes/metastáticas. De salientar, que não ocorre sinalização parácrina entre o endotélio pulmonar e as células tumorais metastáticas, já que não se verifica marcação para N1ICD pulmonar. No caso da avaliação do papel do ganho-de-função endotelial-específico de Dll4, verificouse que a redução do crescimento tumoral resulta duma diminuição da angiogénese (com redução concordante da expressão dos receptores pro-angiogénicos Vgfr1 e Vgfr2), mas também da sua normalização. Trata-se duma árvore vascular com poucas ramificações, com características de maturidade evidenciadas pelo grande revestimento peri-vascular (aumento de células de músculo liso, dos marcadores α-SMA, Pdgfr-βe Ephrin-B2), permitindo um fluxo sanguíneo eficiente, ainda que inferior ao normal, pela redução da densidade vascular. Este efeito, aparentemente paradoxal (contra-intuitivo), acaba por promover um melhor aporte da doxorrubicina (quimioterápico), com bloqueio do crescimento e/ou indução da apoptose tumoral. Esta normalização parece também inibir a metastização, já que apesar de haver um aumento da expressão dos marcadores de EMT, essas células tumorais acabam por ficar “aprisionadas” no endotélio tumoral, dado que se constata uma redução do número de macro-metástases. No entanto, este modelo carece de clarificação no número de células tumorais circulantes, bem como nas restantes etapas da metastização que não chegaram a ser exploradas. Em ambos os modelos, verifica-se que é a função endotelial Dll4/Notch, e não a hipóxia tumoral, que determina a sinalização para EMT e seleção de CSC. Concluindo, observa-se no contexto da dinâmica tumoral fenótipos opostos para os modelos de perda e ganho-de-função endotelial-específico de Dll4, em linha com o que já foi descrito para o contexto da cicatrização de feridas. Estamos novamente perante o conceito de dose-dependência de Dll4, com aparente repercussão também na metastização. Isto porque o fenótipo final é o mesmo, de redução do número de macrometástases, apesar das diferenças na angiogénese tumoral e no evento inicial de EMT, ficando por explorar a restante cascata da metastização. O uso do ligando Dll4, na forma de sub ou sobre-expressão, pode constituir uma nova abordagem terapêutica ao tratamento dos cancros da mama e colo-rectal metastizados, a par de outros anti-angiogénicos já utilizados. Mas mais interessante será explorar a sua abordagem como fármacos antagonistas ou agonistas, numa estratégia de prevenção da metastização.N/

Studies of immunoglobulin receptors and human proximal tubular cells

Glomerulonephritis (GN) is a major cause of progressive chronic renal failure (CRF) and accounts for less than 20% in the UK of patients requiring dialysis and transplantation. There are many forms of GN, of which IgA nephropathy (IgAN) is the most common, affecting up to 2% of the adult population. GN is characterised by irreversible and progressive glomerular and tubulointerstitial fibrosis, reduction in glomerular filtration rate (GFR), and retention of uremic toxins. The rate of progression of GN is increased by various clinical factors including hypertension, and the quantity and specificity of proteinuria. Although albuminuria is most widely studied, higher molecular weight proteins such as immunoglobulins (Ig) are more associated with progression of renal disease. In this thesis I have explored the hypothesis that an interaction between tubular cells and filtered Ig/immuno complex (Ig/IC) may involve specific cellular receptors, and thus provide a novel mechanism for the initiation of tubular injury and fibrosis. Deposition of Ig and IC is seen in renal diseases such as membranous glomerulonephritis, and IgAN. Ig/IC are regarded as responsible for the initiation of glomerular injury in these diseases. However, whether Ig/IC contribute directly to tubular injury is unknown. We previously identified a novel IgA receptor, the Fcα/μ receptor (Fcα/μR), in mesangial cells which may contribute to IgAN. This receptor binds both IgA and IgM but not IgG. I hypothesised that Ig and IC, filtered at the glomerulus, may act on Ig receptors in human proximal tubular epithelial cells (PTEC) and may play a direct pathophysiological role in the development of interstitial fibrosis. Specifically, that filtered IgA may bind to the Fcα/μR in IgAN. The activation of Ig receptors may stimulate tubular cells to alter cell proliferation and extracellular matrix formation (e.g fibronectin (FN)), which are pathophysiological characteristics of tubulointerstitial fibrosis in GN. I studied primary and immortalised human PTEC using qualitative RT-PCR and quantitative real-time PCR (qRT-PCR) for expression of candidate Ig receptors. I investigated the expression of Ig receptors and their regulation by cytokines (e.g. IFN-γ, TGF-β1 and IL-1α) known to be implicated in GN. The receptors studied include the polymeric immunoglobulin receptor (pIgR), the neonatal receptor (FcRn), the Fcα/μR, the classical IgG receptors (FcγR1, γIIa, γIIb, γIII and the related FcR γ-chain) and the classical IgA receptor (FcαR). None of the classical IgG receptors were expressed by PTEC, nor was the classical IgA receptor. However, the FcRn, pIgR and the Fcα/μR were expressed. Gene expression of these receptors was investigated under cytokine regulation. IFN-γ, IL-1α and TGF-β1 had no effect on FcRn gene expression. The expression of the pIgR gene was up-regulated by IFN-γ and the Fcα/μR transcript level was up-regulated by IL-1α and IFN-γ but down-regulated by TGF-β1. Expression of Fcα/µR protein by tubular cells was confirmed by western blot analysis by using specific Fcα/µR monoclonal antibody. Immunohistochemistry confirmed the expression of Fcα/µR by PTEC in both normal and diseased kidney and that IgG, IgA and IgM binding varies in parallel with gene expression of related Fc receptors. This binding was associated with increased FN production (assessed by ELISA) and reduced the proliferation of PTEC, demonstrated by [3H]-thymidine uptake assay. I screened the ability of PTEC to release cytokines under normal conditions and confirmed this by ProteoPlex cytokine array. Among these cytokines there was no significant effect of Ig/IC on IL-6, IL-8 and GM-CSF released by PTEC, and this was confirmed by specific ELISA for each individual cytokine. The phosphotyrosine/phospho-ERK signalling pathway was unaffected by Ig/IC; however, IgM was able to activate the phospho-ERK pathway. These results suggest that there might be alternative signalling pathways activated by Ig/IC in PTEC. I then studied the effect of calcineurin inhibitor immunosuppressive drugs as a model of tubular toxicity, and statins as immunomodulatory drugs, reputed to protect renal function and known to have immunomodulatory effects. I hypothesised that immunosuppressant agents/statins either alone or in combination with LPS might affect Ig receptors expression, cytokine release, FN production and proliferation by PTEC. My results inferred that immunosuppressive agents alone have a non-inflammatory response on Ig receptor expression. However, in combination with LPS, the study drugs showed a slight inhibitory effect on the expression of the pIgR on PTEC at the highest concentration only. There was no significant effect of immunosuppressant agents alone or in combination with LPS on IL-6, IL8 and GM-CSF released by PTEC. Cyclosporine, FK506 and sirolimus were shown to inhibit both the production of FN and the growth of PTEC at high doses, thereby exerting an antiproliferative effect. Both simvastatin and fluvastatin inhibited LPS induced production of IL-6, IL-8 and GM-CSF, FN and proliferation in a dose-dependent manner. 1 micromolar simvastatin/fluvastatin was associated with a 35% reduction in unstimulated FN production, a 51% reduction in proliferation, and reduced production of IL-6 (58%), IL-8 (65%) and GM-CSF (57%). The expression of Ig receptors was increased in a dose-dependent manner in PTEC treated with statins alone or in combination with LPS. In conclusion, the expression of tubular FcRn, pIgR and Fcα/µR by PTEC is regulated by proinflammatory cytokines. The binding of IgA and IgM, possibly through the Fcα/µR, by PTEC may contribute to immune-mediated nephropathy. The FN production and the proliferation suggest that Ig/IC binding may contribute to the pathophysiology of IC-mediated renal disease. The ability of immunosuppressant agents to reduce FN production, proliferation, and pIgR expression by PTEC may offer potential new strategies for the prevention and treatment of nephrotoxicity, proteinuria and chronic allograft nephropathy (CAN). The inhibitory effect of statins alone or in combination with LPS on FN production, proliferation and cytokines released suggest that statin therapy may have potential benefit in interstitial nephritis and fibrosis. On other hand, statins alone or in combination with LPS increased the expression of Ig receptors by PTEC. This thesis shows that the novel human Fcα/µR in PTEC may play an important role in the immune mechanisms involved in the tubular response during renal injury associated with tubular Ig/IC deposits in renal disease

Biochemical and biological effects of irisin in a model of diabetes mellitus

Diabetes mellitus (DM) is a highly prevalent health problem affecting more than 425 million people worldwide. It is associated with several detrimental complications such as neuropathy, nephropathy, retinopathy and cardiovascular diseases. Irisin is a novel hormone that plays a role in metabolism by stimulating the browning of white adipose tissue (WAT) into beige adipose tissue which acquires properties that are similar to those of brown adipose tissue (BAT). Several studies have attempted to characterize the roles of irisin in DM and obesity, however, contradictory results have been reported and physiological roles of irisin have been questioned by several researchers. In our study, we investigated the role of irisin in controlling glucose levels and insulin secretion in STZ-induced DM model and the mechanism by which irisin exerts its beneficial effects both in vivo and in vitro, using a variety of biochemical, morphological and cell biology techniques. We showed that irisin did not cause any significant reduction in weight or fasting blood glucose, however, it caused a significant glucose reduction 30 minutes after glucose challenge. Our data also showed that irisin co-localizes with insulin in pancreatic β-cells in both normal and diabetic animals while it co-localizes with glucagon only in diabetic animals. Moreover, irisin was also detected in skeletal muscle, visceral and subcutaneous adipose tissues. Irisin also reduced triglycerides and increased the level of high density lipoprotein (HDL) and total protein. We also provided evidence that irisin treatment can modulate the tissue level of different peptide hormones such as insulin, glucagon, incretins and leptin. In addition, irisin possesses a potent antioxidant activity and reversed oxidative stress induced by DM. Our in vitro investigations showed that irisin can stimulate the release of insulin from pancreatic β-cells. Irisin could be a potential therapeutic agent in the management of DM

Dissecting the Human Antibody Response to Dengue Virus

Dengue fever (DF) and severe dengue (SD) are two forms of an emerging infectious disease that presents a severe public health crisis predominantly in developing countries. Its etiological agent, Dengue virus (DENV), is a mosquito-borne pathogen, which exists as four different serotypes (named DENV1 through 4). Primary natural infections in humans stimulate a highly cross-reactive antibody response, however, protection is observed to be only against the serotype of infection. Extensive work on the mouse antibody response to DENV has mapped strongly neutralizing antibodies to the domain III (EDIII) of the DENV envelope (E) protein. However, recent work showed that after a natural infection in humans, anti-EDIII antibodies contribute very little to protection. Therefore, the human memory antibody response against a natural DENV infection remains poorly understood. Our present studies characterized both circulating polyclonal antibodies from human sera and human monoclonal antibodies from memory B-cells, after late convalescent primary DENV infections. The present body of work shows that after late convalescent natural primary DENV infections, humans produce two uniquely different antibody groups: 1) A cross-reactive, weakly neutralizing group that makes up the dominant proportion of anti-DENV antibodies, and 2) a minor group of strongly neutralizing, type-specific antibodies. Subsequently, we mapped some of these strongly neutralizing type-specific antibodies to a novel complex, quaternary epitope that includes the hinge region between domains I and II (EDI-II) of the E protein. Due to the role of the EDI-DII hinge region in DENV fusion and entry, this epitope offers a functional advantage. As hypothesized, we observed that all neutralizing EDI-DII hinge-binding monoclonal antibodies that were isolated blocked DENV infection at a step post-attachment, while the mechanism of neutralization by human polyclonal sera was more variable. In parallel, we found that the weakly neutralizing, cross-reactive group of antibodies was responsible for antibody-mediated enhancement of infection by heterotypic DENV serotypes. Further investigation mapped these enhancing cross-reactive antibodies to the DENV surface glycoproteins prM and E protein. These studies shed some light on the protective and enhancing DENV epitopes targeted by the human immune response, and set the stage for a safer and efficacious human vaccine against DENV.Doctor of Philosoph