Hypochlorous acid (HOCl) is a powerful oxidant produced by activated phagocytes at sites of inflammation to kill a wide range of pathogens. Yet, it may also damage and kill the neighbouring host cells. The abundance of dead macrophages in atherosclerotic plaques and their colocalization with HOCl-modified proteins implicate HOCl may play a role in killing macrophages, contributing to disease progression. The first part of this research was to investigate the cytotoxic effect and cell death mechanism(s) of HOCl on macrophages. Macrophages require efficient defense mechanism(s) against HOCl to function properly at inflammatory sites. The second part of the thesis was to examine the antioxidative effects of glutathione (GSH) and 7,8-dihydroneopterin (7,8-NP) on HOCl-induced cellular damage in macrophages. GSH is an efficient scavenger of HOCl and a major intracellular antioxidant against oxidative stress, whereas 7,8-NP is secreted by human macrophages upon interferon-γ (IFN-γ) induction during inflammation and can also scavenge HOCl. HOCl caused concentration-dependent cell viability loss in human monocyte derived macrophage (HMDM) cells above a specific concentration threshold. HOCl reacted with HMDMs to cause viability loss within the first 10 minutes of treatment, and it posed no latent effect on the cells afterwards regardless of the HOCl concentrations. The lack of caspase-3 activation, rapid influx of propidium iodide (PI) dye, rapid loss of intracellular ATP and cell morphological changes (cell swelling, cell membrane integrity loss and rupture) were observed in HMDM cells treated with HOCl. These results indicate that HOCl caused HMDM cells to undergo necrotic cell death. In addition to the loss of intracellular ATP, HOCl also caused rapid loss of GAPDH enzymatic activity and mitochondrial membrane potential, indicating impairment of the metabolic energy production. Loss of the mitochondrial membrane potential was mediated by mitochondrial permeability transition (MPT), as blocking MPT pore formation using cyclosporin A (CSA) prevented mitochondrial membrane potential loss. HOCl caused an increase in cytosolic calcium ion (Ca2+) level, which was due to both intra- and extra-cellular sources. However, extracellular sources only contributed significantly above a certain HOCl concentration. Preventing cytosolic Ca2+ increase significantly inhibited HOCl-induced cell viability loss. This suggests that cytosolic Ca2+ increase was associated with HOCl-induced necrotic cell death in HMDM cells, possibly via the activation of Ca2+-dependent calpain cysteine proteases. Calpain inhibitors prevented HOCl-induced lysosomal destabilisation and cell viability loss in HMDM cells. Calpains induced HOCl-induced necrotic cell death possibly by degrading cytoskeletal and other cellular proteins, or causing the release of cathepsin proteases from ruptured lysosomes that also degraded cellular components. The HOCl-induced cytosolic Ca2+ increase also caused mitochondrial Ca2+ accumulation and MPT activation-mediated mitochondrial membrane potential loss. MPT activation, like calpain activation, was also associated with the HOCl-induced necrotic cell death, as preventing MPT activation completely inhibited HOCl-induced cell viability loss. The involvement of both calpain activation and MPT activation in HOCl-induced necrotic cell death in HMDM cells implies a cause and effect relationship between these two events. HMDM cells depleted of intracellular GSH using diethyl maleate showed increased susceptibility towards HOCl insult compared to HMDM cells with intact intracellular GSH levels, indicating that intracellular GSH played an important role in protecting HMDM cells against HOCl exposure. Intracellular GSH level in each HMDM cell preparation directly correlated with HOCl concentration required to kill 50% of population for each cell preparation, indicating intracellular GSH concentrations determine the efficiency of GSH in preventing HOCl-induced damage to HMDM cells. Intracellular GSH and cell viability loss induced by 400 μM HOCl were significantly prevented by 300 μM extracellular 7,8-NP, indicating that added 7,8-NP is an efficient scavenger of HOCl and out-competed intracellular GSH for HOCl. The amount of 7,8-NP synthesized by HMDM cells upon IFN-γ induction was too low to efficiently prevent HOCl-mediated intracellular GSH and cell viability loss. HOCl clearly causes HMDM cells to undergo necrosis when the concentration exceeds the intracellular GSH concentrations. Above this concentration HOCl causes oxidative damage to the Ca2+ ion channels on cell and ER membranes, resulting in an influx of Ca2+ ions into the cytosol and possibly the mitochondria. The rise in Ca2+ ions triggers calpain activation, resulting in the MPT-mediated loss of mitochondrial membrane potential, lysosomal instability and cellular necrosis.

Les coxsackievirus du groupe B (CVB) sont des petits virus à ARN appartenant à au genre Enterovirus et à la famille des Picornaviridae. Chez, l’homme, les CVB sont responsables de nombreuses infections aiguës bénignes ou sévères. Ils sont également incriminés dans le développement de maladies chroniques telles que le diabète de type 1 (DT1). En effet, plusieurs données épidémio-cliniques sont en faveur d’un lien entre les entérovirus et notamment les CVB et le DT1. Deux mécanismes majeurs ont été proposés pour expliquer cette pathogenèse entérovirale du DT1. Il s’agit de l’activation « en passant » d’un environnement inflammatoire et la persistance virale qui concourent à l’initiation du processus auto-immun. Les études présentées dans cette thèse visent à comprendre les caractéristiques et conséquences de l’infection à CVB qui pourraient expliquer l’implication de ces mécanismes. Les résultats obtenus suggèrent que CVB4 (utilisé comme modèle des CVB) est un virus inflammatoire. In vitro, il induit la production de grandes quantités d’IFNα par les cellules mononuclées du sang (CMN). Néanmoins cette induction d’IFNα n’est possible qu’en cas de facilitation de l’infection par des anticorps non neutralisants, qui se traduit par une entrée importante du virus dans les cellules. Dans nos travaux, l’IFNα a été détecté dans le plasma de sujets diabétiques, et fréquemment associé à la présence d’ARN entéroviral. De même, parmi les CMN, les monocytes ont été identifiés comme les principales cellules cibles du virus. En dehors de l’IFNα, nous avons montré que CVB4 peut induire la synthèse de plusieurs autres cytokines pro-inflammatoires notamment l’IL-6 et le TNFα. De façon intéressante, l’infection des cellules n’est pas indispensable car cette induction est possible par des particules non infectieuses. Cette production de cytokines pro-inflammatoires par les CMN peut également être amplifiée par la facilitation de l’infection en présence de particules infectieuses de CVB4. Nous avons montré que les macrophages, cellules effectrices importantes de l’immunité innée au niveau tissulaire, peuvent produire en présence de CVB4 de l’IFNα et d’autres cytokines pro-inflammatoires. Les macrophages dérivés de CMN en présence de M-CSF (mais pas de GM-CSF) sont infectables par CVB4 et le virus peut persister dans ces cellules. CVB4 peut également établir une infection chronique productive de type « état porteur » dans des cellules canalaires pancréatiques. Les cellules chroniquement infectées peuvent être guéries grâce à un traitement par de la fluoxétine. Cette molécule utilisée dans le traitement de troubles psychiatriques, présente in vitro une activité antivirale vis-à-vis de certains entérovirus, et permet d’éliminer complètement en quelques semaines le virus des cellules chroniquement infectées par CVB4. Des modifications cellulaires ont été observées au niveau des cellules chroniquement infectées notamment une diminution de l’expression de PDX-1, une résistance à la lyse au cours d’une réinfection par CVB4, ainsi qu’une diminution très importante de l’expression du récepteur CAR. Ces modifications cellulaires acquises au cours de l’infection chronique pouvaient persister après l’élimination du virus. Les cellules chroniquement infectées présentent également un profil de microARNs très différent de celui des cellules non infectées. Une évolution du virus a été également observée avec des changements phénotypiques et génotypiques. L’ensemble de nos observations montre que les caractéristiques de l’infection à CVB4 sont compatibles avec les mécanismes évoqués dans la pathogenèse entérovirale du DT1 et renforcent l’hypothèse de l’implication des CVB dans cette maladie. / Group B coxsackieviruses (CVB) are small RNA viruses belonging to Enterovirus genus and to the Picornaviridae family. In humans, CVB can cause numerous mild and severe acute infections. They are also thought to be involved in the development of chronic diseases such as type 1 diabetes (T1D). Several epidemiological and clinical data support a link between enteroviruses, especially CVB and T1D. Two main mechanisms have been described to explain this enteroviral pathogenesis of T1D including a “bystander activation” of an inflammatory environment and viral persistence. These mechanisms contribute to initiation of the autoimmune process. Our studies aimed to understand the features and outcomes of CVB infection that could explain their involvement in these mechanisms. The results suggest that CVB4 (used as CVB model) is an inflammatory virus. CVB4 induces in vitro the production by peripheral blood mononuclear cells (PBMCs) of high amounts of IFNα. However this induction is only possible when CVB4 infection is enhanced by non-neutralizing antibodies, resulting in increased viral entry in cells. We also reported detection of IFNα in plasma of T1D patients, commonly associated with enteroviral RNA. In addition, monocytes have been identified as major targets of enteroviruses among PBMCs. Besides IFNα, CVB4 can induce the synthesis of other proinflammatory cytokines, mainly IL-6 and TNFα. Interestingly, infection is not needed, since inactivated viral particles can induce these proinflammatory cytokines. In addition, the enhancing of CVB4 infection in PBMCs results in increased production of these cytokines. We have shown that macrophages that are known as major innate immunity effectors can produce IFNα and other proinflammatory cytokines upon infection with CVB4. Macrophages derived from PBMCs in presence of M-CSF (but not GM-CSF) can be infected by CVB4, and the virus can persist in these cells. CVB4 can also establish a productive, carrier-sate persistent infection in pancreatic ductal-like cells. The virus can be completely cleared from chronically-infected cells using fluoxetine. This molecule already used in the treatment of depression and other mental disorders, has displayed antiviral activity against many enteroviruses, and can completely clear CVB4 from chronically-infected cells within few weeks. Cellular changes have been observed during chronic infection including a reduced expression of PDX-1, a resistant profile to lysis upon superinfection with CVB4, and an important decrease of CAR expression. These changes can linger even after the clearance of CVB4. In addition the miRNA profile in chronically-infected ductal-like cells was clearly different from that of mock-infected cells. Some phenotypic and genotypic changes were also observed in the virus derived from chronic infection. Altogether, these findings show the features of CVB4 infection are compatible with mechanisms reported in the enteroviral pathogenesis of T1D, and support the hypothesis of involvement of CVB in this disease.

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.

Os venenos de serpentes contêm concentrações elevadas de fosfolipases A2 secretadas (sFLA2), que apresentam homologia com as FLA2s de mamíferos, cujos níveis estão aumentados em doenças inflamatórias. Neste estudo, investigou-se a ativação e a expressão de fatores envolvidos na formação de corpúsculos lipídicos (CLs) em células fagociticas e o papel desses fatores na resposta imune inata, induzida pela MT-III, uma sFLA2s de veneno. A MT-III induziu aumento dos níveis de triacilglicerol, colesterol e lisofosfolipideos e a ativação e expressão dos fatores PPAR-g, PPAR-d/b, SREBP2 e do CD36. Sob estimulo da MT-III, o receptor PPAR-b/d, as enzimas DGAT, ACAT e FAS foram relevantes para a formação de CLs e para a expressão da PLIN2. O CD36 participa da expressão da COX-2, sem modificar a liberação de PGE2. O TLR2 e a MyD88 foram essenciais para a formação de CLs e síntese da IL-1b e IL-10. Ainda, o TLR2 foi relevante para a liberação de PGE2, PGD2 e LTB4, enquanto MyD88 foi fundamental somente para a liberação de PGE2 e expressão da PLIN2, induzidas pela MT-III. / Snake venoms contain high concentrations of secreted phospholipase A2 (sPLA2) with homology to mammalian PLA2s, whose levels are elevated in inflammatory diseases. In this study, we investigated activation and expression of factors involved in lipid droplets formation (LDs) and participation that factors in the innate immune response induced by MT-III, sPLA2s from snake venom, in phagocytic cells. MT-III induced increase of triacylglycerol, cholesterol and lysophospholipids levels and activation and expression of factors PPAR-g, PPAR-d/b, SREBP2 and CD36. PPAR-b/d receptor, DGAT, ACAT and FAS enzymes were relevant to LDs formation and critical to PLIN2 expression induced by MT-III. CD36 participates in COX-2 expression without modifying PGE2 release stimulated by MT-III. TLR2 and MyD88 were essential to LDs formation and IL-1b and IL-10 synthesis stimulated by MT-III. Moreover, TLR2 was relevant to PGE2, PGD2 and LTB4 biosynthesis, while MyD88 is essential only for PGE2 release and PLIN2 expression induced by MT-III.

IL-12 family cytokines IL-23 and IL-27 play an important role linking innate and adaptive immunity, and regulating T-cell responses. The production of IL-12, a structurally similar cytokine, is decreased in chronic HIV infection; therefore IL-23 and IL-27 may also be influenced by HIV infection. I hypothesized that HIV inhibits LPS-induced IL-23 and IL-27 production in human MDMs by suppressing the activation of signalling pathways regulating their expression. In vitro HIV-infection of MDMs did not have any effect on basal secretion of IL-23 or IL-27; however, HIV inhibited LPS-induced production of IL-12/23 p40 and IL-23 p19, and IL-27 EBI3 and IL-27 p28 mRNA expression, and IL-23, IL-12/23 p40 and IL-27 secretion. In order to evaluate the molecular mechanisms by which HIV inhibits IL-23 and IL-27 in LPS-stimulated MDMs, the signalling pathways regulating their expression were evaluated. The PI3K, p38 MAPK, and JNK MAPK pathways were found to positively regulate LPS-induced IL-27 secretion. Interestingly, in vitro HIV infection inhibited LPS-induced p38 and JNK MAPK activation in MDMs. In summary, I have shown that HIV inhibits IL-23 and IL-27 production in LPS-stimulated MDMs and that HIV may inhibit LPS-induced IL-27 production through the inhibition of p38 and JNK MAPK activation. It is currently unknown whether PKCs regulate LPS-induced IL-23 or IL-27 in human monocytes/macrophages. I demonstrated that classical PKCs differentially regulate LPS-induced IL-23 and IL-27 secretion within THP-1 cells, primary monocytes, and MDMs. Classical PKCs were found to positively regulate LPS-induced IL-12/23 p40 and IL-27 p28 mRNA expression and IL-12/23 p40, IL-23, and IL-27 secretion in primary human monocytes. Similarly, the classical PKCs were found to positively regulate IL-27 p28 mRNA expression and IL-27 secretion in THP-1 cells. However, classical PKCs did not regulate LPS-induced IL-27 production in MDMs, or LPS-induced IL-23 production in THP-1 cells. Overall, this demonstrates that classical PKCs differentially regulate LPS-induced IL-23 and IL-27 production in different myeloid cells.

Introduction: Extracellular DNA (ecDNA) is a common component of blood plasma. Increased levels of ecDNA in plasma can be found in some autoimmune diseases like systemic lupus erythematosus (SLE), rheumatoid arthritis or celiac disease which are associated with inflammatory processes. These diseases are also associated with an increased risk of osteoporosis. Bone is a dynamic structure undergoing constant modelling caused by osteoblasts, osteocytes and osteoclasts. Shifting their equilibrium can lead to pathological conditions such as osteoporosis. In this thesis we focused on elucidating whether ecDNA, an inflammatory agent with proven immunoregulatory effects can alter differentiation potential of monocytes and alternatively lead to osteoclastogenesis via TLR9. Material and methods: We obtained monocytes from peripheral blood of healthy donors and cultivated them with four types of ODNs control (CO), stimulatory (ST), inhibitory (INH, telomeric (TLM) with phosphodiester (-pO) or phosphorothioate (-pS) backbone for two weeks to establish their effect on differentiation potential of monocytes into osteoclasts. Osteoclastogenesis was evaluated by number of yielded osteoclasts observed on a light microscope. To establish the effect of ODNs on osteoclast activity samples were analysed by qPCR for...

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.

Human mast cells are often found perivascularly and at mucosal sites and may play crucial roles in the inflammatory response. Recent studies have suggested a prominent role for mast cells in host defense. In this study, we analyzed the effects of a common airway pathogen, Moraxella catarrhalis and a commensal bacterium, Neiserria cinerea, on activation of human mast cells. Human mast cell leukemia cells (HMC-1) were activated with either phorbol myristate acetate (PMA) and calcium ionophore or with varying concentrations of heat-killed suspensions of bacteria. Supernatants were assayed for the cytokines interleukin-4 (IL-4), granulocyte macrophage colony stimulating factor (GM-CSF), IL-6, IL-8, IL-13 and monocyte chemotactic protein-1 (MCP-1). Nuclear proteins were isolated and assayed by electrophoretic mobility shift assay (EMSA) for nuclear factor kappaB (NF-κB) nuclear binding activity. In some experiments, NF-κB inhibitor, Bay-11 was added to determine functional significance. Both M. catarrhalis and N. cinerea induced mast cell activation and selective secretion of two key inflammatory cytokines, IL-6 and MCP-1. This was accompanied by NF-κB activation. Neither spun bacterial supernatants nor bacterial lipopolysaccharide induced cytokine secretion, suggesting need for direct bacterial contact with mast cells. Scanning electron microscopy revealed active aggregation of bacteria over mast cell surfaces. The NF-κB inhibitor, Bay-11, inhibited expression of MCP-1. These findings suggest the possibility of direct interactions between human mast cells and common bacteria and provide evidence for a novel role for human mast cells in innate immunity.

Hepatitis C virus (HCV) induces a high rate of chronic infection via dysregulation of host immunity. We have previously shown that T-cell immunoglobulin and mucin domain protein-3 (Tim-3) is up-regulated on monocyte/macrophages (M/Mφ) during chronic HCV infection; little is known, however, about the transcription factor that controls its expression in these cells. In this study, we investigated the role of transcription factor, T-box expressed in T cells (T-bet), in Tim-3 expression in M/Mφ in the setting of HCV infection. We demonstrate that T-bet is constitutively expressed in resting CD14+ M/Mφ in the peripheral blood. M/Mφ from chronically HCV-infected individuals exhibit a significant increase in T-bet expression that positively correlates with an increased level of Tim-3 expression. Up-regulation of T-bet is also observed in CD14+ M/Mφ incubated with HCV+ Huh7.5 cells, as well as in primary M/Mφ or monocytic THP-1 cells exposed to HCV core protein in vitro, which is reversible by blocking HCV core/gC1qR interactions. Moreover, the HCV core-induced up-regulation of T-bet and Tim-3 expression in M/Mφ can be abrogated by incubating the cells with SP600125 – an inhibitor for the c-Jun N-terminal kinase (JNK) signalling pathway. Importantly, silencing T-bet gene expression decreases Tim-3 expression and enhances interleukin-12 secretion as well as signal transducer and activator of transcription 1 phosphorylation. These data suggest that T-bet, induced by the HCV core/gC1qR interaction, enhances Tim-3 expression via the JNK pathway, leading to dampened M/Mφ function during HCV infection. These findings reveal a novel mechanism for Tim-3 regulation via T-bet during HCV infection, providing new targets to combat this global epidemic viral disease.

Coronary heart disease, an inflammatory disease, is the leading cause of death globally. White blood cell counts (including monocytes) are easily available biomarkers of systemic inflammation. Monocyte subtypes can be measured by flow cytometry and classified into classical (CD14high, CD16neg), intermediate (CD14high, CD16+) and non-classical (CD14+, CD16high) with distinct functional properties. The goal of this study was to investigate the association of monocyte total count and its subtypes with cardiovascular risk groups defined by the Framingham Risk Score, which is used to estimate the 10-year risk of developing myocardial infarction or predict mortality following coronary heart disease. We also aimed to investigate whether monocyte counts are associated with relevant cardiovascular risk factors not included in the Framingham Risk Score, such as carotid atherosclerotic plaque and intima-media thickness. Our data came from the LIFE-Adult study, a population-based cohort study of 10,000 randomly selected participants in Leipzig, Germany. Data was gathered using self-administered questionnaires and physical examinations. Carotid plaques and intima-media thickness were measured using carotid artery sonography. Monocyte subtypes in blood were determined by 10-color flow cytometry for a total of 690 individuals. In a multivariate regression analysis adjusting for the risk factors BMI, intima-media thickness, presence of carotid plaques and diabetes mellitus, monocyte subtypes and total count were found to be significantly associated with the dichotomized Framingham Risk Score (≥10% versus <10%): Odds ratios [95% confidence interval] for monocyte subtypes: classical: 11.19 [3.79–34.26]; intermediate: 2.27 [1.11–4.71]; non-classical: 4.18 [1.75–10.20]; total: 14.59 [4.61–47.95]. In absence of prospective data, the FRS was used as a surrogate for CHD. Our results indicate that monocyte counts could provide useful predictive value for cardiovascular disease risk.