Belal Chami

Therapeutic Inflammatory Monocyte Modulation Using Immune-Modifying Microparticles

Negatively charged immune-modifying microparticles bind to the scavenger receptor MARCO on inflammatory monocytes, resulting in their apoptosis and reduced inflammatory damage in a range of diseases. A New Frontier in Immune Modulation Inflammatory monocytes markedly potentiate the immune pathology observed in many diseases, yet no therapy exists that specifically inhibits these cells. The therapeutic accessibility of monocytes in the bloodstream and their inherent propensity to engulf particulate material suggest that highly negatively charged microparticles might provide a readily translatable solution to this problem. These microparticles, referred to as immune-modifying microparticles (IMPs), may be derived from numerous compounds, including the biodegradable polymer poly(lactic-co-glycolic acid) (PLGA-IMP), already used in humans for inter alia dissolvable sutures. Getts et al. now show that upon infusion, IMPs bind to a receptor with a positive domain on inflammatory monocytes, resulting in monocyte sequestration in the spleen and apoptosis through a similar pathway observed for senescing leukocytes. This safe monocyte clearance pathway culminated in substantially reduced inflammatory tissue damage in mouse models of West Nile virus encephalitis, experimental autoimmune encephalomyelitis, peritonitis, colitis, and myocardial infarction. Together, the data suggest that IMPs could transform the treatment of acute inflammation. Indeed, phase 1/2 testing is planned to begin in 2014, with rapid translation supported by the availability of clinical-grade PLGA. Inflammatory monocyte-derived effector cells play an important role in the pathogenesis of numerous inflammatory diseases. However, no treatment option exists that is capable of modulating these cells specifically. We show that infused negatively charged, immune-modifying microparticles (IMPs), derived from polystyrene, microdiamonds, or biodegradable poly(lactic-co-glycolic) acid, were taken up by inflammatory monocytes, in an opsonin-independent fashion, via the macrophage receptor with collagenous structure (MARCO). Subsequently, these monocytes no longer trafficked to sites of inflammation; rather, IMP infusion caused their sequestration in the spleen through apoptotic cell clearance mechanisms and, ultimately, caspase-3–mediated apoptosis. Administration of IMPs in mouse models of myocardial infarction, experimental autoimmune encephalomyelitis, dextran sodium sulfate–induced colitis, thioglycollate-induced peritonitis, and lethal flavivirus encephalitis markedly reduced monocyte accumulation at inflammatory foci, reduced disease symptoms, and promoted tissue repair. Together, these data highlight the intricate interplay between scavenger receptors, the spleen, and inflammatory monocyte function and support the translation of IMPs for therapeutic use in diseases caused or potentiated by inflammatory monocytes.

Oxidative stress in Alzheimer's disease: Primary villain or physiological by-product?

Abstract The prevalence of Alzheimers disease (AD) is increasing rapidly worldwide due to an ageing population and largely ineffective treatments. In AD cognitive decline is due to progressive neuron loss that begins in the medial temporal lobe and spreads through many brain regions. Despite intense research the pathogenesis of the common sporadic form of AD remains largely unknown. The popular amyloid cascade hypothesis suggests that the accumulation of soluble oligomers of beta amyloid peptides (Aβ) initiates a series of events that cause neuronal loss. Among their putative toxic effects, Aβ oligomers are thought to act as pro-oxidants combining with redox-active metals to produce excessive reactive oxygen and nitrogen species. However, to date the experimental therapies that reduce Aβ load in AD have failed to halt cognitive decline. Another hypothesis proposed by the late Mark Smith and colleagues is that oxidative stress, rather than Aβ, precipitates the pathogenesis of AD. That is, Aβ and microtubule-associated protein tau are upregulated to address the redox imbalance in the AD brain. As the disease progresses, excess Aβ and tau oligomerise to further accelerate the disease process. Here, we discuss redox balance in the human brain and how this balance is affected by ageing. We then discuss where oxidative stress is most likely to act in the disease process and the potential for intervention to reduce its effects.

The Role of CXCR3 in DSS-Induced Colitis

Inflammatory bowel disease (IBD) is a group of disorders that are characterized by chronic, uncontrolled inflammation in the intestinal mucosa. Although the aetiopathogenesis is poorly understood, it is widely believed that IBD stems from a dysregulated immune response towards otherwise harmless commensal bacteria. Chemokines induce and enhance inflammation through their involvement in cellular trafficking. Reducing or limiting the influx of these proinflammatory cells has previously been demonstrated to attenuate inflammation. CXCR3, a chemokine receptor in the CXC family that binds to CXCL9, CXCL10 and CXCL11, is strongly overexpressed in the intestinal mucosa of IBD patients. We hypothesised that CXCR3 KO mice would have impaired cellular trafficking, thereby reducing the inflammatory insult by proinflammatory cell and attenuating the course of colitis. To investigate the role of CXCR3 in the progression of colitis, the development of dextran sulfate sodium (DSS)-induced colitis was investigated in CXCR3−/− mice over 9 days. This study demonstrated attenuated DSS-induced colitis in CXCR3−/− mice at both the macroscopic and microscopic level. Reduced colitis correlated with lower recruitment of neutrophils (p = 0.0018), as well as decreased production of IL-6 (p<0.0001), TNF (p = 0.0038), and IFN-γ (p = 0.0478). Overall, our results suggest that CXCR3 plays an important role in recruiting proinflammatory cells to the colon during colitis and that CXCR3 may be a therapeutic target to reduce the influx of proinflammatory cells in the inflamed colon.

The rise and fall of insulin signaling in Alzheimer's disease.

The prevalence of both diabetes and Alzheimer’s disease (AD) are reaching epidemic proportions worldwide. Alarmingly, diabetes is also a risk factor for Alzheimer’s disease. The AD brain is characterised by the accumulation of peptides called Aβ as plaques in the neuropil and hyperphosphorylated tau protein in the form of neurofibrillary tangles within neurons. How diabetes confers risk is unknown but a simple linear relationship has been proposed whereby the hyperinsulinemia associated with type 2 diabetes leads to decreased insulin signaling in the brain, with downregulation of the PI3K/AKT signalling pathway and its inhibition of the major tau kinase, glycogen synthase kinase 3β. The earliest studies of post mortem AD brain tissue largely confirmed this cascade of events but subsequent studies have generally found either an upregulation of AKT activity, or that the relationship between insulin signaling and AD is independent of glycogen synthase kinase 3β altogether. Given the lack of success of beta-amyloid-reducing therapies in clinical trials, there is intense interest in finding alternative or adjunctive therapeutic targets for AD. Insulin signaling is a neuroprotective pathway and represents an attractive therapeutic option. However, this incredibly complex signaling pathway is not fully understood in the human brain and particularly in the context of AD. Here, we review the ups and downs of the research efforts aimed at understanding how diabetes modifies AD risk.

Pre-treatment with the synthetic antioxidant T-butyl bisphenol protects cerebral tissues from experimental ischemia reperfusion injury

Treatments to inhibit or repair neuronal cell damage sustained during focal ischemia/reperfusion injury in stroke are largely unavailable. We demonstrate that dietary supplementation with the antioxidant di‐tert‐butyl‐bisphenol (BP) before injury decreases infarction and vascular complications in experimental stroke in an animal model. We confirm that BP, a synthetic polyphenol with superior radical‐scavenging activity than vitamin E, crosses the blood–brain barrier and accumulates in rat brain. Supplementation with BP did not affect blood pressure or endogenous vitamin E levels in plasma or cerebral tissue. Pre‐treatment with BP significantly lowered lipid, protein and thiol oxidation and decreased infarct size in animals subjected to middle cerebral artery occlusion (2 h) and reperfusion (24 h) injury. This neuroprotective action was accompanied by down‐regulation of hypoxia inducible factor‐1α and glucose transporter‐1 mRNA levels, maintenance of neuronal tissue ATP concentration and inhibition of pro‐apoptotic factors that together enhanced cerebral tissue viability after injury. That pre‐treatment with BP ameliorates oxidative damage and preserves cerebral tissue during focal ischemic insult indicates that oxidative stress plays at least some causal role in promoting tissue damage in experimental stroke. The data strongly suggest that inhibition of oxidative stress through BP scavenging free radicals in vivo contributes significantly to neuroprotection.

Serum Amyloid A Receptor Blockade and Incorporation into High-Density Lipoprotein Modulates Its Pro-Inflammatory and Pro-Thrombotic Activities on Vascular Endothelial Cells

The acute phase protein serum amyloid A (SAA), a marker of inflammation, induces expression of pro-inflammatory and pro-thrombotic mediators including ICAM-1, VCAM-1, IL-6, IL-8, MCP-1 and tissue factor (TF) in both monocytes/macrophages and endothelial cells, and induces endothelial dysfunction—a precursor to atherosclerosis. In this study, we determined the effect of pharmacological inhibition of known SAA receptors on pro-inflammatory and pro-thrombotic activities of SAA in human carotid artery endothelial cells (HCtAEC). HCtAEC were pre-treated with inhibitors of formyl peptide receptor-like-1 (FPRL-1), WRW4; receptor for advanced glycation-endproducts (RAGE), (endogenous secretory RAGE; esRAGE) and toll-like receptors-2/4 (TLR2/4) (OxPapC), before stimulation by added SAA. Inhibitor activity was also compared to high-density lipoprotein (HDL), a known inhibitor of SAA-induced effects on endothelial cells. SAA significantly increased gene expression of TF, NFκB and TNF and protein levels of TF and VEGF in HCtAEC. These effects were inhibited to variable extents by WRW4, esRAGE and OxPapC either alone or in combination, suggesting involvement of endothelial cell SAA receptors in pro-atherogenic gene expression. In contrast, HDL consistently showed the greatest inhibitory action, and often abrogated SAA-mediated responses. Increasing HDL levels relative to circulating free SAA may prevent SAA-mediated endothelial dysfunction and ameliorate atherogenesis.

CXCR3 plays a critical role for host protection against Salmonellosis

CXCR3 and its ligands are heavily associated with inflammation and have been implicated in numerous inflammatory diseases. CXCR3 plays an important role in recruiting pro-inflammatory cells, specifically neutrophils, in a model of sterile colitis whereby CXCR3−/− mice showed an attenuated course of colitis with markedly reduced host-tissue damage in the inflamed caecum. The role of CXCR3 during infectious colitis, however, is unclear and therefore in this study, we investigated the role of CXCR3 in the regulation of the immune response during acute and chronic gastrointestinal infection, using a murine model of Salmonella enterica serovar Enteritidis. During acute infection with Salmonella, we observed significantly increased Salmonella loading in the caecum and dissemination to the spleen and liver in CXCR3−/− mice, but not in Wt counterparts. During chronic infection, increased pathological features of inflammation were noted in the spleen and liver, with significantly increased levels of apoptosis in the liver of CXCR3−/− mice, when compared to Wt counterparts. In addition, compromised intestinal IgA levels, CD4+ helper T cells and neutrophil recruitment were observed in CXCR3−/− challenged with Salmonella, when compared to Wt counterparts. Our data suggests that CXCR3 is a key molecule in host intestinal immunity against Salmonellosis via regulating neutrophils chemotaxis.

Evidence supporting oxidative stress in a moderately affected area of the brain in Alzheimer’s disease

The pathogenesis of Alzheimer’s disease (AD) remains to be elucidated. Oxidative damage and excessive beta-amyloid oligomers are components of disease progression but it is unclear how these factors are temporally related. At post mortem, the superior temporal gyrus (STG) of AD cases contains plaques, but displays few tangles and only moderate neuronal loss. The STG at post mortem may represent a brain region that is in the early stages of AD or alternately a region resistant to AD pathogenesis. We evaluated expression profiles and activity of endogenous anti-oxidants, oxidative damage and caspase activity in the STG of apolipoprotein ε4-matched human AD cases and controls. Total superoxide dismutase (SOD) activity was increased, whereas total glutathione peroxidase (GPX), catalase (CAT) and peroxiredoxin (Prx) activities, were decreased in the AD-STG, suggesting that hydrogen peroxide accumulates in this brain region. Transcripts of the transcription factor NFE2L2 and inducible HMOX1, were also increased in the AD-STG, and this corresponded to increased Nuclear factor erythroid 2-related factor (NRF-2) and total heme-oxygenase (HO) activity. The protein oxidation marker 4-hydroxynonenal (4-HNE), remained unchanged in the AD-STG. Similarly, caspase activity was unaltered, suggesting that subtle redox imbalances in early to moderate stages of AD do not impact STG viability.

Molecular changes to the rat renal cotransporters PEPT1 and PEPT2 due to ageing

Renal PEPT1 and PEPT2 cotransporters play an important role in the balance of circulating body oligopeptides and selected peptidomimetic drugs. We aim to comprehensively characterise age-related changes of the renal PEPT cotransporters at the gene, protein, and functional level. Brush border membrane vesicles (BBMV) and outer medulla membrane vesicles (OMMV) were isolated from the kidneys of young, middle-aged and old rats. The protein expression of PEPT1 was not only increased in BBMV from old rats, but PEPT1 also appeared in OMMV from middle-aged and old rats. SLC15A1 gene expression in the renal cortex increased in middle-aged group. PEPT2 protein expression was not only increased with ageing, but PEPT2 also was found in BBMV from middle-aged and old groups. SLC15A2 gene expression in the renal outer medulla increased in the old group. These changes in the expressions and localisations of PEPT1 and PEPT2 could explain the changes to transport activity in BBMV and OMMV. These findings provide novel insights that would be useful for maintaining protein nutrition and optimising the delivery of some peptidomimetic drugs in elderly individuals.

Myeloperoxidase in the inflamed colon: A novel target for treating inflammatory bowel disease

Inflammatory bowel disease (IBD) is a debilitating disorder involving inflammation of the gastrointestinal tract. The incidence of IBD is increasing worldwide. Immunological responses in the gastrointestinal (GI) tract to altered gut microbiota, mucosal injury and loss of intestinal epithelial cell function all contribute to a complex mechanism underlying IBD pathogenesis. Immune cell infiltration, particularly neutrophils, is a histological feature of IBD. This innate immune response is aimed at resolving intestinal damage however, neutrophils and monocytes that are recruited and accumulate in the GI wall, participate in IBD pathogenesis by producing inflammatory cytokines and soluble mediators such as reactive oxygen species (ROS; one- and two-electron oxidants). Unregulated ROS production in host tissue is linked to oxidative damage and inflammation and may potentiate mucosal injury. Neutrophil-myeloperoxidase (MPO) is an abundant granule enzyme that catalyses production of potent ROS; biomarkers of oxidative damage (and MPO protein) are increased in the mucosa of patients with IBD. Targeting MPO may mitigate oxidative damage to host tissue and ensuing inflammation. Here we identify mechanisms by which MPO activity perpetuates inflammation and contributes to host-tissue injury in patients with IBD and discuss MPO as a potential therapeutic target to protect the colon from inflammatory injury.