Oral administration of the KATP channel opener diazoxide ameliorates disease progression in a murine model of multiple sclerosis

Background Multiple Sclerosis (MS) is an acquired inflammatory demyelinating disorder of the central nervous system (CNS) and is the leading cause of nontraumatic disability among young adults. Activated microglial cells are important effectors of demyelination and neurodegeneration, by secreting cytokines and others neurotoxic agents. Previous studies have demonstrated that microglia expresses ATP-sensitive potassium (KATP) channels and its pharmacological activation can provide neuroprotective and anti-inflammatory effects. In this study, we have examined the effect of oral administration of KATP channel opener diazoxide on induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Methods Anti-inflammatory effects of diazoxide were studied on lipopolysaccharide (LPS) and interferon gamma (IFNy)-activated microglial cells. EAE was induced in C57BL/6J mice by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Mice were orally treated daily with diazoxide or vehicle for 15 days from the day of EAE symptom onset. Treatment starting at the same time as immunization was also assayed. Clinical signs of EAE were monitored and histological studies were performed to analyze tissue damage, demyelination, glial reactivity, axonal loss, neuronal preservation and lymphocyte infiltration. Results Diazoxide inhibited in vitro nitric oxide (NO), tumor necrosis factor alpha (TNF-¿) and interleukin-6 (IL-6) production and inducible nitric oxide synthase (iNOS) expression by activated microglia without affecting cyclooxygenase-2 (COX-2) expression and phagocytosis. Oral treatment of mice with diazoxide ameliorated EAE clinical signs but did not prevent disease. Histological analysis demonstrated that diazoxide elicited a significant reduction in myelin and axonal loss accompanied by a decrease in glial activation and neuronal damage. Diazoxide did not affect the number of infiltrating lymphocytes positive for CD3 and CD20 in the spinal cord. Conclusion Taken together, these results demonstrate novel actions of diazoxide as an anti-inflammatory agent, which might contribute to its beneficial effects on EAE through neuroprotection. Treatment with this widely used and well-tolerated drug may be a useful therapeutic intervention in ameliorating MS disease.

Antigen binding properties of purified immunoglobulin A and reconstituted secretory immunoglobulin A antibodies.

The hybridoma cell line ZAC3 expresses Vibrio cholerae lipopolysaccharide (LPS)-specific mouse IgA molecules as a heterogeneous population of monomeric (IgAm), dimeric (IgAd), and polymeric (IgAp) forms. We describe a gentle method combining ultrafiltration, ion-exchange chromatography, and size exclusion chromatography for the simultaneous and qualitative separation of the three molecular forms. Milligram quantities of purified IgA molecules were recovered allowing for direct comparison of the biological properties of the three forms. LPS binding specificity was tested after purification; IgAd and IgAp were found to bind strongly to LPS whereas IgAm did not. Secretory IgA (sIgA) could be reconstituted in vitro by combining recombinant secretory component (rSC) and purified IgAd or IgAp, but not IgAm. Surface plasmon resonance-based binding experiments using LPS monolayers indicated that purified reconstituted sIgA and IgA molecules recognize LPS with identical affinity (KA 1.0 x 10(8)M-1). Thus, this very sensitive assay provides the first evidence that the function of SC in sIgA complex is not to modify the affinity for the antigen. KA falls to 6.6 x 10(5) M-1 when measured by calorimetry using detergent-solubilized LPS and IgA, suggesting that the LPS environment is critical for recognition by the antibody.

New formulation of vasoactive intestinal peptide using liposomes in hyaluronic acid gel for uveitis.

We evaluated the benefits of a novel formulation of vasoactive intestinal peptide (VIP) based on the incorporation of VIP-loaded rhodamine-conjugated liposomes (VIP-Rh-Lip) within hyaluronic acid (HA) gel (Gel-VIP-Rh-Lip) for the treatment of endotoxin-induced uveitis (EIU) in comparison with VIP-Rh-Lip alone. In vitro release study and rheological analysis showed that interactions between HA chains and liposomes resulted in increased viscosity and reinforced elasticity of the gel. In vivo a single intravitreal injection of Gel-VIP-Rh-Lip was performed in rats 7 days prior to uveitis induction by subcutaneous lipopolysaccharide injection. The maximal ocular inflammation occurs within 16-24 h in controls (VIP-Rh-Lip, unloaded-Rh-Lip). Whereas intraocular injection of VIP-Rh-Lip had no effect on EIU severity compared with controls, Gel-VIP-Rh-Lip reduced significantly the clinical score and number of inflammatory cells infiltrating the eye. The fate of liposomes, VIP and HA in the eyes, regional and inguinal lymph nodes and spleen was analyzed by immunostaining and fluorescence microscopy. Retention of liposomes by HA gel was observed in vitro and in vivo. Inflammation severity seemed to impact on system stability resulting in the delayed release of VIP. Thus, HA gel containing VIP-Rh-Lip is an efficient strategy to obtain a sustained delivery of VIP in ocular and lymph node tissues.

Activated B cells express functional Fas ligand.

Fas ligand (FasL, Apo-1L) is a member of the tumor necrosis factor protein family and binding to its receptor (Fas, Apo-1, CD95) triggers cell death through apoptosis. Ligand expression is restricted to cells with known cytolytic activity and found on hematopoietic cells of the T cell and natural killer lineage. Here we provide evidence that B lymphocytes can express FasL. Flow cytometric analysis revealed that FasL is expressed on the surface of B cells upon stimulation with either lipopolysaccharide or phorbol 12-myristate 13-acetate/ionomycin. FasL expression on activated B cells was confirmed by western blot and reverse transcriptase polymerase chain reaction analysis. FasL on B cells is functional since lipopolysaccharide-activated B lymphocytes derived from wild type, but not from gld mutant mice, were able to kill Fas-sensitive target cells. Our data suggest that the Fas system may contribute to the control of B cell homeostasis.

Effects of the PPAR-beta agonist GW501516 in an in vitro model of brain inflammation and antibody-induced demyelination.

BACKGROUND: Brain inflammation plays a central role in numerous brain pathologies, including multiple sclerosis (MS). Microglial cells and astrocytes are the effector cells of neuroinflammation. They can be activated also by agents such as interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). Peroxisome proliferator-associated receptor (PPAR) pathways are involved in the control of the inflammatory processes, and PPAR-beta seems to play an important role in the regulation of central inflammation. In addition, PPAR-beta agonists were shown to have trophic effects on oligodendrocytes in vitro, and to confer partial protection in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. In the present work, a three-dimensional brain cell culture system was used as in vitro model to study antibody-induced demyelination and inflammatory responses. GW 501516, a specific PPAR-beta agonist, was examined for its capacity to protect from antibody-mediated demyelination and to prevent inflammatory responses induced by IFN-gamma and LPS. METHODS: Aggregating brain cells cultures were prepared from embryonal rat brain, and used to study the inflammatory responses triggered by IFN-gamma and LPS and by antibody-mediated demyelination induced by antibodies directed against myelin-oligodendrocyte glycoprotein (MOG). The effects of GW 501516 on cellular responses were characterized by the quantification of the mRNA expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), inducible NO synthase (i-NOS), PPAR-beta, PPAR-gamma, glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), and high molecular weight neurofilament protein (NF-H). GFAP expression was also examined by immunocytochemistry, and microglial cells were visualized by isolectin B4 (IB4) and ED1 labeling. RESULTS: GW 501516 decreased the IFN-gamma-induced up-regulation of TNF-alpha and iNOS in accord with the proposed anti-inflammatory effects of this PPAR-beta agonist. However, it increased IL-6 m-RNA expression. In demyelinating cultures, reactivity of both microglial cells and astrocytes was observed, while the expression of the inflammatory cytokines and iNOS remained unaffected. Furthermore, GW 501516 did not protect against the demyelination-induced changes in gene expression. CONCLUSION: Although GW 501516 showed anti-inflammatory activity, it did not protect against antibody-mediated demyelination. This suggests that the protective effects of PPAR-beta agonists observed in vivo can be attributed to their anti-inflammatory properties rather than to a direct protective or trophic effect on oligodendrocytes.

Effects of neuroinflammation on demyelination and remyelination: an in vitro study in aggregating brain cell cultures

La neuroinflammation joue un rôle important dans de nombreuses maladies neurodégéneratives dont la sclérose en plaques. Les microglies et les astrocytes sont les cellules effectrices de la réponse inflammatoire dans le cerveau et sont impliquées dans les processus de démyélinisation et de remyélinisation. Dans ce travail, nous avons étudié les réactions inflammatoires accompagnant la démyélinisation et leurs conséquences sur la remyélinisation. Dans ce but, trois différents traitements démyélinisants ont été appliqués sur des cultures en agrégats de télencéphales de rats, à savoir (i) la lysophosphatidylcholine, (ii) l'interféron-γ (IFN-γ) combiné avec du lipopolysaccharide (LPS), et (iii) des anticorps dirigés contre la MOG (myelin oligodendrocyte glycoprotein) en présence de complément. Nous avons montré que ces traitements induisent différents types de démyélinisation, de réponses inflammatoires et d'effets secondaires sur les neurones. Nous avons ensuite examiné les effets de l'atténuation de la réponse inflammatoire sur la démyélinisation et la remyélinisation, en utilisant la minocycline, un antibiotique bloquant la réactivité microgiale, et le GW 5501516, un agoniste de PPAR-β (peroxisome proliferator-activated receptor-β). Nous avons montré que la minocycline prévient l'activation microgliale induite par le traitement avec l'IFN-γ et le LPS, mais qu'elle ne protège pas de la démyélinisation. Néanmoins, elle induit une remyélinisation, probablement en favorisant la maturation d'oligodendrocytes immatures. Le GW 501516 diminue l'expression de l'IFN-γ après une démyélinisation induite par les anticorps anti-MOG, mais il ne prévient pas la démyélinisation et ne favorise pas la remyélinisation. Ces résultats indiquent que la démyélinisation induite par le traitement avec l'IFN-γ et le LPS n'est pas une conséquence directe de l'activation microgliale, et que l'augmentation de l'expression de l'IFN-γ ne participe pas à la démyélinisation induite par les anticorps anti-MOG. Ces résultats suggèrent que l'atténuation de l'activation microgliale est bénéfique pour la remyélinisation.

Structural investigations into the interaction of hemoglobin and part structures with bacterial endotoxins.

An understanding of details of the interaction mechanisms of bacterial endotoxins (lipopolysaccharide, LPS) with the oxygen transport protein hemoglobin is still lacking, despite its high biological relevance. Here, a biophysical investigation into the endotoxin:hemoglobin interaction is presented which comprises the use of various rough mutant LPS as well as free lipid A; in addition to the complete hemoglobin molecule from fetal sheep extract, also the partial structure alpha-chain and the heme-free sample are studied. The investigations comprise the determination of the gel-to-liquid crystalline phase behaviour of the acyl chains of LPS, the ultrastructure (type of aggregate structure and morphology) of the endotoxins, and the incorporation of the hemoglobins into artificial immune cell membranes and into LPS. Our data suggest a model for the interaction between Hb and LPS in which hemoglobins do not react strongly with the hydrophilic or with the hydrophobic moiety of LPS, but with the complete endotoxin aggregate. Hb is able to incorporate into LPS with the longitudinal direction parallel to the lipid A double-layer. Although this does not lead to a strong disturbance of the LPS acyl chain packing, the change of the curvature leads to a slightly conical molecular shape with a change of the three-dimensional arrangement from unilamellar into cubic LPS aggregates. Our previous results show that cubic LPS structures exhibit strong endotoxic activity. The property of Hb on the physical state of LPS described here may explain the observation of an increase in LPS-mediating endotoxicity due to the action of Hb.

Probing the interaction between feline immunodeficiency virus and CD134 by using the novel monoclonal antibody 7D6 and the CD134 (Ox40) ligand.

The feline immunodeficiency virus (FIV) targets activated CD4-positive helper T cells preferentially, inducing an AIDS-like immunodeficiency in its natural host species, the domestic cat. The primary receptor for FIV is CD134, a member of the tumor necrosis factor receptor superfamily, and all primary viral strains tested to date use CD134 for infection. We examined the expression of CD134 in the cat using a novel anti-feline CD134 monoclonal antibody (MAb), 7D6, and showed that as in rats and humans, CD134 expression is restricted tightly to CD4+, and not CD8+, T cells, consistent with the selective targeting of these cells by FIV. However, FIV is also macrophage tropic, and in chronic infection the viral tropism broadens to include B cells and CD8+ T cells. Using 7D6, we revealed CD134 expression on a B220-positive (B-cell) population and on cultured macrophages but not peripheral blood monocytes. Moreover, macrophage CD134 expression and FIV infection were enhanced by activation in response to bacterial lipopolysaccharide. Consistent with CD134 expression on human and murine T cells, feline CD134 was abundant on mitogen-stimulated CD4+ T cells, with weaker expression on CD8+ T cells, concordant with the expansion of FIV into CD8+ T cells with progression of the infection. The interaction between FIV and CD134 was probed using MAb 7D6 and soluble CD134 ligand (CD134L), revealing strain-specific differences in sensitivity to both 7D6 and CD134L. Infection with isolates such as PPR and B2542 was inhibited well by both 7D6 and CD134L, suggesting a lower affinity of interaction. In contrast, GL8, CPG, and NCSU were relatively refractory to inhibition by both 7D6 and CD134L and, accordingly, may have a higher-affinity interaction with CD134, permitting infection of cells where CD134 levels are limiting.

Protection from lethal Gram-negative bacterial sepsis by targeting Toll-like receptor 4

Rapport de synthèse : L'immunité innée regroupe les mécanismes moléculaires et cellulaires formant la première ligne de défense contre les infections microbiennes. La détection des micro-organismes pathogènes est assurée par des cellules sentinelles (cellules dendritiques et macrophages) qui jouent un rôle fondamental dans l'initiation des mécanismes de défense de l'hôte. Au contact de produits microbiens, ces cellules produisent un large échantillonnage de molécules, dont des cytokines, impliquées dans le développement de la réponse inflammatoire. La régulation de cette réponse relève d'un équilibre délicat, son insuffisance tant que son excès pouvant compromettre le devenir des patients infectés. La sepsis sévère et le choc septique représentent les formes les plus sévères d'infection, et leur mortalité demeure élevée (25 à 30% pour la sepsis sévère et 50 à 60% pour le choc septique). De plus, l'incidence de la sepsis tend à augmenter, atteignant en 2000 plus de 240 cas pour 100'000 personnes en Grande-Bretagne. La sepsis est caractérisée dans sa phase aiguë par une réponse inflammatoire exubérante. La plupart des thérapies visant à la bloquer ont toutefois montré des bénéfices incertains lors de leur application clinique. Il est donc impératif d'identifier de nouvelles cibles thérapeutiques. Les "Toll-like receptors" (TLRs) sont une famille de récepteurs qui jouent un rôle fondamental dans la détection des micro-organismes par les cellules du système immunitaire inné. Parmi eux, TLR4 est indispensable à la reconnaissance du lipopolysaccharide (LPS) des bactéries Gram-négatives. L'interaction entre TLR4 et le LPS représentant un élément précoce de la réponse de l'hôte à l'infection, nous avons émit l'hypothèse que TLR4 pourrait représenter une cible de choix en vue du développement de nouvelles thérapies contre la sepsis. Dans l'objectif de valider ce concept, nous avons, dans un premier temps, démontré que des souris génétiquement déficientes en TLR4 étaient totalement résistantes au choc septique induit par Escherichia coli (E. coli), une bactérie Gram-négative fréquemment responsable de sepsis. Forts de cette observation, nous avons développé une molécule recombinante composée du domaine extracellulaire de TLR4 fusionné à la partie IgGi-Fc. Cette molécule soluble, qui inhibait la réponse des macrophages au LPS in vitro, a été utilisée pour générer des anticorps anti-TLR4 chez le lapin. La spécificité et l'efficacité de ces anticorps ont été prouvées en démontrant que les anti-TLR4 bloquaient les signaux d'activation intracellulaire et la production de TNF et d'IL-6 en réponse au LPS et aux bactéries Gram-négatives in vitro et in vivo. Enfin, l'efficacité des ces anticorps a été testée dans des modèles de sepsis chez la souris. Ainsi, l'injection prophylactique (-lh) ou thérapeutique (+3h) d'anticorps anti-TLR4 réduisait la production de TNF et protégeait les animaux de la mort. De manière spectaculaire, ces anticorps réduisaient également la production de TNF et protégeaient de la sepsis à E. coli lorsqu'ils étaient administrés de manière prophylactique (-4h) et thérapeutique, jusqu'à 13 heures après l'initiation de l'infection. Ces résultats indiquent donc qu'il est possible de bloquer le développement de la réponse inflammatoire et de protéger du choc septique à bactéries Gram-négatives en utilisant des thérapies ciblant TLR4. Par ailleurs, ils suggèrent qu'une fenêtre d'opportunité de plusieurs heures pourrait être mise à profit pour initier un traitement chez les patients septiques. Ces résultats devraient encourager la poursuite des essais cliniques en cours qui visent à tester l'efficacité de thérapies dirigées contre TLR4 comme traitement complémentaire de la sepsis.

Plasminogen activator inhibitor-1 activity and 4G/5G polymorphism in hemodialysis

Introduction: Chronic insufficiency alters homeostasis, in part due to endothelial inflammation. Plasminogen activator inhibitor-1 (PAI-1) is increased in renal disease, contributing to vascular damage. We assessed PAI-1 activity and PAI-1 4G/5G polymorphism in hemodialysis (HD) subjects and any association between thrombotic vascular access (VA) events and PAI-1 polymorphism. Methods: Prospective, observational study in 36 HD patients: mean age: 66.6 +/- 12.5 yr, males n=26 (72%), time on HD: 28.71 +/- 22.45 months. Vascular accesses: 10 polytetrafluoroethylene grafts (PTFEG), 22 arteriovenous fistulae (AVF), four dual lumen catheters (CAT). Control group (CG): 40 subjects; mean age: 60.0 +/- 15 yrs, males n=30 (75%). Group A (GA): thrombotic events (n=12), and group B (GB): No events (n=24). Groups were no different according to age (69.2 +/- 9.12 vs. 65.3 +/- 14.5 yrs), gender (males: 7; 58.3% vs. 18; 81.8%), time on HD (26.1 +/- 14.7 vs. 30.1 +/- 38.7 months), causes of renal failure. Time to follow-up, for access thrombosis: 12 months. Results: PAI-1 levels in HD: 7.21 +/- 2.13 vs. CG: 0.42 +/- 0.27 U/ml (p < 0.000 1). PAI-1 4G/5G polymorphic variant distribution in HD: 5G/5G: 6 (17%),4G/5G: 23 (64%); 4G/4G: 7 (19%) and in CG: 5G/5G: 14 (35%); 4G/5G: 18 (45%); 4G/4G: 8 (20%). C-reactive protein (CRP) in HD: 24.5 +/- 15.2 mg/L vs. in CG 2.3 +/- 0.2 mg/L (p < 0.0001). PAI-1 4G/5G variants: GA: 5G/5G: 3; 4G/5G: 8; 4G/4G: 1; GB: 5G/5G: 3; 4G/5G: 15; 4G/4G: 6. Thrombosis occurred in 8/10 patients (80%) with PTFEG, 3/22 (9%) in AVF, and 1/4 (25%) in CAT. Among the eight PTFEG patients with thrombosis, seven were PAI 4G/5G. Conclusions: PAI-1 levels were elevated in HD patients, independent of their polymorphic variants, 4G/5G being the most prevalent variant. Our data suggest that in patients with PTFEG the 4G/5G variant might be associated with an increased thrombosis risk.

Cellular and molecular evidence for a role of tumor necrosis factor alpha in the ovulatory mechanism of trout

Background: The relevance of immune-endocrine interactions to the regulation of ovarian function in teleosts is virtually unexplored. As part of the innate immune response during infection, a number of cytokines such as tumor necrosis factor alpha (TNF alpha) and other immune factors, are produced and act on the reproductive system. However, TNF alpha is also an important physiological player in the ovulatory process in mammals. In the present study, we have examined for the first time the effects of TNF alpha in vitro in preovulatory ovarian follicles of a teleost fish, the brown trout (Salmo trutta). Methods: To determine the in vivo regulation of TNF alpha expression in the ovary, preovulatory brook trout (Salvelinus fontinalis) were injected intraperitoneally with either saline or bacterial lipopolysaccharide (LPS). In control and recombinant trout TNF alpha (rtTNF alpha)-treated brown trout granulosa cells, we examined the percentage of apoptosis by flow cytometry analysis and cell viability by propidium iodide (PI) staining. Furthermore, we determined the in vitro effects of rtTNF alpha on follicle contraction and testosterone production in preovulatory brown trout ovarian follicles. In addition, we analyzed the gene expression profiles of control and rtTNF alpha-treated ovarian tissue by microarray and real-time PCR (qPCR) analyses. Results: LPS administration in vivo causes a significant induction of the ovarian expression of TNF alpha. Treatment with rtTNF alpha induces granulosa cell apoptosis, decreases granulosa cell viability and stimulates the expression of genes known to be involved in the normal ovulatory process in trout. In addition, rtTNF alpha causes a significant increase in follicle contraction and testosterone production. Also, using a salmonid-specific microarray platform (SFA2.0 immunochip) we observed that rtTNF alpha induces the expression of genes known to be involved in inflammation, proteolysis and tissue remodeling. Furthermore, the expression of kallikrein, TOP-2, serine protease 23 and ADAM 22, genes that have been postulated to be involved in proteolytic and tissue remodeling processes during ovulation in trout, increases in follicles incubated in the presence of rtTNF alpha. Conclusions In view of these results, we propose that TNF alpha could have an important role in the biomechanics of follicle weakening, ovarian rupture and oocyte expulsion during ovulation in trout, primarily through its stimulation of follicular cell apoptosis and the expression of genes involved in follicle wall proteolysis and contraction.

Arginase II Promotes Macrophage Inflammatory Responses Through Mitochondrial Reactive Oxygen Species, Contributing to Insulin Resistance and Atherogenesis.

BACKGROUND: Macrophage-mediated chronic inflammation is mechanistically linked to insulin resistance and atherosclerosis. Although arginase I is considered antiinflammatory, the role of arginase II (Arg-II) in macrophage function remains elusive. This study characterizes the role of Arg-II in macrophage inflammatory responses and its impact on obesity-linked type II diabetes mellitus and atherosclerosis. METHODS AND RESULTS: In human monocytes, silencing Arg-II decreases the monocytes' adhesion to endothelial cells and their production of proinflammatory mediators stimulated by oxidized low-density lipoprotein or lipopolysaccharides, as evaluated by real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Macrophages differentiated from bone marrow cells of Arg-II-deficient (Arg-II(-/-)) mice express lower levels of lipopolysaccharide-induced proinflammatory mediators than do macrophages of wild-type mice. Importantly, reintroducing Arg-II cDNA into Arg-II(-/-) macrophages restores the inflammatory responses, with concomitant enhancement of mitochondrial reactive oxygen species. Scavenging of reactive oxygen species by N-acetylcysteine prevents the Arg-II-mediated inflammatory responses. Moreover, high-fat diet-induced infiltration of macrophages in various organs and expression of proinflammatory cytokines in adipose tissue are blunted in Arg-II(-/-) mice. Accordingly, Arg-II(-/-) mice reveal lower fasting blood glucose and improved glucose tolerance and insulin sensitivity. Furthermore, apolipoprotein E (ApoE)-deficient mice with Arg-II deficiency (ApoE(-/-)Arg-II(-/-)) display reduced lesion size with characteristics of stable plaques, such as decreased macrophage inflammation and necrotic core. In vivo adoptive transfer experiments reveal that fewer donor ApoE(-/-)Arg-II(-/-) than ApoE(-/-)Arg-II(+/+) monocytes infiltrate into the plaque of ApoE(-/-)Arg-II(+/+) mice. Conversely, recipient ApoE(-/-)Arg-II(-/-) mice accumulate fewer donor monocytes than do recipient ApoE(-/-)Arg-II(+/+) animals. CONCLUSIONS: Arg-II promotes macrophage proinflammatory responses through mitochondrial reactive oxygen species, contributing to insulin resistance and atherogenesis. Targeting Arg-II represents a potential therapeutic strategy in type II diabetes mellitus and atherosclerosis. (J Am Heart Assoc. 2012;1:e000992 doi: 10.1161/JAHA.112.000992.).

Microglia, Calcification and Neurodegenerative Diseases

Neurodegeneration is a complex process involving different cell types andneurotransmitters. A common characteristic of neurodegenerative disorders such asAlzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis, Huntington’s disease (HD) and Amyotrophic Lateral Sclerosis (ALS) is the occurrence of a neuroinflammatoryreaction in which cellular processes involving glial cells (mainly microglia and astrocytes) and T cells are activated in response to neuronal death. This inflammatory reaction has recently received attention as an unexpected potential target for the treatment of these diseases.Microglial cells have a mesenchymal origin, invade the central nervous system (CNS)prenatally (Chan et al., 2007b) and are the resident macrophages in the CNS (Ransohoff &Perry, 2009). They comprise approximately 10-20% of adult glia and serve as the CNS innateimmune system. In neurodegenerative diseases, microglia is activated by misfoldedproteins. In the case of AD, amyloid- (A ) peptides accumulate extracellularly and activate the microglia locally. In the case of PD, ALS and HD, the misfolded proteins accumulate intracellularly but are still associated with activation of the microglia (Perry et al., 2010). Reactive microglia in the substantia nigra and striatum of PD brains have been described, and increased levels of proinflammatory cytokines and inducible nitric oxide synthase havebeen detected in these brain regions, providing evidence of a local inflammatory reaction (Hirsch & Hunot, 2009). The injection of lipopolysaccharide (a potent microglia activator) into the substantia nigra produces microglial activation and the death of dopaminergic cells. These findings support the hypothesis that microglial activation and neuroinflammationcontribute to PD pathogenesis (Herrera et al., 2000)...

Proinflammatory activity of cell-wall constituents from gram-positive bacteria.

Innate immunity reacts to conserved bacterial molecules. The outermost lipopolysaccharide (LPS) of Gram-negative organisms is highly inflammatory. It activates responsive cells via specific CD14 and toll-like receptor-4 (TLR4) surface receptor and co-receptors. Gram-positive bacteria do not contain LPS, but carry surface teichoic acids, lipoteichoic acids and peptidoglycan instead. Among these, the thick peptidoglycan is the most conserved. It also triggers cytokine release via CD14, but uses the TLR2 co-receptor instead of TLR4 used by LPS. Moreover, whole peptidoglycan is 1000-fold less active than LPS in a weight-to-weight ratio. This suggests either that it is not important for inflammation, or that only part of it is reactive while the rest acts as ballast. Biochemical dissection of Staphylococcus aureus and Streptococcus pneumoniae cell walls indicates that the second assumption is correct. Long, soluble peptidoglycan chains (approximately 125 kDa) are poorly active. Hydrolysing these chains to their minimal unit (2 sugars and a stem peptide) completely abrogates inflammation. Enzymatic dissection of the pneumococcal wall generated a mixture of highly active fragments, constituted of trimeric stem peptides, and poorly active fragments, constituted of simple monomers and dimers or highly polymerized structures. Hence, the optimal constraint for activation might be 3 cross-linked stem peptides. The importance of structural constraint was demonstrated in additional studies. For example, replacing the first L-alanine in the stem peptide with a D-alanine totally abrogated inflammation in experimental meningitis. Likewise, modifying the D-alanine decorations of lipoteichoic acids with L-alanine, or deacylating them from their diacylglycerol lipid anchor also decreased the inflammatory response. Thus, although considered as a broad-spectrum pattern-recognizing system, innate immunity can detect very subtle differences in Gram-positive walls. This high specificity underlines the importance of using well-characterized microbial material in investigating the system.

Pheomelanin-based coloration and the ability to cope with variation in food supply and parasitism.

Although gene by environment interactions may play a key role in the maintenance of genetic polymorphisms, little is known about the ecological factors involved in these interactions. We investigated whether food supply and parasites can mediate covariation between the degree of adult pheomelanin-based coloration, a heritable trait, and offspring body mass in the tawny owl (Strix aluco). We swapped clutches between nests to allocate genotypes randomly among environments. Three weeks after hatching, we challenged the immune system of 80 unrelated nestlings with either a phytohemagglutinin (PHA) or a lipopolysaccharide, surrogates of alternative parasites, and then fed them ad lib. or food-restricted them during the following 6 days in the laboratory. Whatever the immune challenge, nestlings fed ad lib. converted food more efficiently into body mass when their biological mother was dark pheomelanic. In contrast, food-restricted nestlings challenged with PHA lost less body mass when their biological mother was pale pheomelanic. Nestling tawny owls born from differently melanic mothers thus show differing reaction norms relative to food availability and parasitism. This suggests that dark and pale pheomelanic owls reflect alternative adaptations to food availability and parasites, factors known to vary in space and time.