AB5384 Sigma-AldrichAnti-Nitric Oxide Synthase II Antibody

Inducible nitric oxide synthase (iNOS) activity increases in acute and chronic inflammatory lung diseases. Imaging iNOS expression may be useful as an inflammation biomarker for monitoring lung disease activity. We developed a novel tracer for PET that binds to iNOS in vivo, (18)F-NOS. In this study, we tested whether (18)F-NOS could quantify iNOS expression from endotoxin-induced lung inflammation in healthy volunteers.Healthy volunteers were screened to exclude cardiopulmonary disease. Qualifying volunteers underwent a baseline, 1-h dynamic (18)F-NOS PET/CT scan. Endotoxin (4 ng/kg) was then instilled bronchoscopically in the right middle lobe. (18)F-NOS imaging was performed again approximately 16 h after endotoxin instillation. Radiolabeled metabolites were determined from blood samples. Cells recovered by bronchoalveolar lavage (BAL) after imaging were stained immunohistochemically for iNOS. (18)F-NOS uptake was quantified as the distribution volume ratio (DVR) determined by Logan plot graphical analysis in volumes of interest placed over the area of endotoxin instillation and in an equivalent lung region on the left. The mean Hounsfield units (HUs) were also computed using the same volumes of interest to measure density changes.Seven healthy volunteers with normal pulmonary function completed the study with evaluable data. The DVR increased by approximately 30%, from a baseline mean of 0.42 ± 0.07 to 0.54 ± 0.12, and the mean HUs by 11% after endotoxin in 6 volunteers who had positive iNOS staining in BAL cells. The DVR did not change in the left lung after endotoxin. In 1 volunteer with low-level iNOS staining in BAL cells, the mean HUs increased by 7% without an increase in DVR. Metabolism was rapid, with approximately 50% of the parent compound at 5 min and 17% at 60 min after injection.(18)F-NOS can be used to image iNOS activity in acute lung inflammation in humans and may be a useful PET tracer for imaging iNOS expression in inflammatory lung disease.

In multiple sclerosis (MS), diffuse degenerative processes in the deep grey matter have been associated with clinical disabilities. We performed a systematic study in MS deep grey matter with a focus on the incidence and topographical distribution of lesions in relation to white matter and cortex in a total sample of 75 MS autopsy patients and 12 controls. In addition, detailed analyses of inflammation, acute axonal injury, iron deposition and oxidative stress were performed. MS deep grey matter was affected by two different processes: the formation of focal demyelinating lesions and diffuse neurodegeneration. Deep grey matter demyelination was most prominent in the caudate nucleus and hypothalamus and could already be seen in early MS stages. Lesions developed on the background of inflammation. Deep grey matter inflammation was intermediate between low inflammatory cortical lesions and active white matter lesions. Demyelination and neurodegeneration were associated with oxidative injury. Iron was stored primarily within oligodendrocytes and myelin fibres and released upon demyelination. In addition to focal demyelinated plaques, the MS deep grey matter also showed diffuse and global neurodegeneration. This was reflected by a global reduction of neuronal density, the presence of acutely injured axons, and the accumulation of oxidised phospholipids and DNA in neurons, oligodendrocytes and axons. Neurodegeneration was associated with T cell infiltration, expression of inducible nitric oxide synthase in microglia and profound accumulation of iron. Thus, both focal lesions as well as diffuse neurodegeneration in the deep grey matter appeared to contribute to the neurological disabilities of MS patients.

Recent data suggest that oxidative injury may play an important role in demyelination and neurodegeneration in multiple sclerosis (MS). We compared the extent of oxidative injury in MS lesions with that in experimental models driven by different inflammatory mechanisms. It was only in a model of coronavirus-induced demyelinating encephalomyelitis that we detected an accumulation of oxidised phospholipids, which was comparable in extent to that in MS. In both, MS and coronavirus-induced encephalomyelitis, this was associated with massive microglial and macrophage activation, accompanied by the expression of the NADPH oxidase subunit p22phox but only sparse expression of inducible nitric oxide synthase (iNOS). Acute and chronic CD4(+) T cell-mediated experimental autoimmune encephalomyelitis lesions showed transient expression of p22phox and iNOS associated with inflammation. Macrophages in chronic lesions of antibody-mediated demyelinating encephalomyelitis showed lysosomal activity but very little p22phox or iNOS expressions. Active inflammatory demyelinating lesions induced by CD8(+) T cells or by innate immunity showed macrophage and microglial activation together with the expression of p22phox, but low or absent iNOS reactivity. We corroborated the differences between acute CD4(+) T cell-mediated experimental autoimmune encephalomyelitis and acute MS lesions via gene expression studies. Furthermore, age-dependent iron accumulation and lesion-associated iron liberation, as occurring in the human brain, were only minor in rodent brains. Our study shows that oxidative injury and its triggering mechanisms diverge in different models of rodent central nervous system inflammation. The amplification of oxidative injury, which has been suggested in MS, is only reflected to a limited degree in the studied rodent models.

Cortical lesions constitute an important part of multiple sclerosis pathology. Although inflammation appears to play a role in their formation, the mechanisms leading to demyelination and neurodegeneration are poorly understood. We aimed to identify some of these mechanisms by combining gene expression studies with neuropathological analysis. In our study, we showed that the combination of inflammation, plaque-like primary demyelination and neurodegeneration in the cortex is specific for multiple sclerosis and is not seen in other chronic inflammatory diseases mediated by CD8-positive T cells (Rasmussen's encephalitis), B cells (B cell lymphoma) or complex chronic inflammation (tuberculous meningitis, luetic meningitis or chronic purulent meningitis). In addition, we performed genome-wide microarray analysis comparing micro-dissected active cortical multiple sclerosis lesions with those of tuberculous meningitis (inflammatory control), Alzheimer's disease (neurodegenerative control) and with cortices of age-matched controls. More than 80% of the identified multiple sclerosis-specific genes were related to T cell-mediated inflammation, microglia activation, oxidative injury, DNA damage and repair, remyelination and regenerative processes. Finally, we confirmed by immunohistochemistry that oxidative damage in cortical multiple sclerosis lesions is associated with oligodendrocyte and neuronal injury, the latter also affecting axons and dendrites. Our study provides new insights into the complex mechanisms of neurodegeneration and regeneration in the cortex of patients with multiple sclerosis.

Sporadic inclusion body myositis is a severely disabling myopathy. The design of effective treatment strategies is hampered by insufficient understanding of the complex disease pathology. Particularly, the nature of interrelationships between inflammatory and degenerative pathomechanisms in sporadic inclusion body myositis has remained elusive. In Alzheimer's dementia, accumulation of β-amyloid has been shown to be associated with upregulation of nitric oxide. Using quantitative polymerase chain reaction, an overexpression of inducible nitric oxide synthase was observed in five out of ten patients with sporadic inclusion body myositis, two of eleven with dermatomyositis, three of eight with polymyositis, two of nine with muscular dystrophy and two of ten non-myopathic controls. Immunohistochemistry confirmed protein expression of inducible nitric oxide synthase and demonstrated intracellular nitration of tyrosine, an indicator for intra-fibre production of nitric oxide, in sporadic inclusion body myositis muscle samples, but much less in dermatomyositis or polymyositis, hardly in dystrophic muscle and not in non-myopathic controls. Using fluorescent double-labelling immunohistochemistry, a significant co-localization was observed in sporadic inclusion body myositis muscle between β-amyloid, thioflavine-S and nitrotyrosine. In primary cultures of human myotubes and in myoblasts, exposure to interleukin-1β in combination with interferon-γ induced a robust upregulation of inducible nitric oxide synthase messenger RNA. Using fluorescent detectors of reactive oxygen species and nitric oxide, dichlorofluorescein and diaminofluorescein, respectively, flow cytometry revealed that interleukin-1β combined with interferon-γ induced intracellular production of nitric oxide, which was associated with necrotic cell death in muscle cells. Intracellular nitration of tyrosine was noted, which partly co-localized with amyloid precursor protein, but not with desmin. Pharmacological inhibition of inducible nitric oxide synthase by 1400W reduced intracellular production of nitric oxide and prevented accumulation of β-amyloid, nitration of tyrosine as well as cell death inflicted by interleukin-1β combined with interferon-γ. Collectively, these data suggest that, in skeletal muscle, inducible nitric oxide synthase is a central component of interactions between interleukin-1β and β-amyloid, two of the most relevant molecules in sporadic inclusion body myositis. The data further our understanding of the pathology of sporadic inclusion body myositis and may point to novel treatment strategies.

Damage to axons in acute multiple sclerosis (MS) lesions is now well established but the mechanisms of this damage remain obscure. Here we have applied a panel of antibodies that identify cell populations and proteins contained in them with a view to detecting those cells and proteins that are localised particularly closely to damaged axons in acute, sub-acute and border-active MS plaques. Results are expressed semi-quantitatively and graphs produced that show that many of the markers show enhanced expression at sites of axon damage. However, the sharpest increase in expression in relation to axon damage was seen for Calpain I (micro-calpain), inducible nitric oxide synthase and MMP-2, suggesting that these proteins may form part of a group of proteins responsible for the initiation of myelin and/or axon damage seen in MS lesions.

To study the expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) and their role in inflammatory bowel disease (IBD).We examined the effect of sera obtained from patients with active Crohn's disease (CD) and ulcerative colitis (UC) on the function and viability of human umbilical vein endothelial cells (HUVEC). HUVECs were cultured for 0-48 h in the presence of a medium containing pooled serum of healthy controls, or serum from patients with active CD or UC. Expression of eNOS and iNOS was visualized by immunofluorescence, and quantified by the densitometry of Western blots. Proliferation activity was assessed by computerized image analyses of Ki-67 immunoreactive cells, and also tested in the presence of the NOS inhibitor, 10(-4) mol/L L-NAME. Apoptosis and necrosis was examined by the annexin-V-biotin method and by propidium iodide staining, respectively.In HUVEC immediately after exposure to UC, serum eNOS was markedly induced, reaching a peak at 12 h. In contrast, a decrease in eNOS was observed after incubation with CD sera and the eNOS level was minimal at 20 h compared to control (18%+/-16% vs 23%+/-15% Pless than 0.01). UC or CD serum caused a significant increase in iNOS compared to control (UC: 300%+/-21%; CD: 275%+/-27% vs 108%+/-14%, Pless than 0.01). Apoptosis/necrosis characteristics did not differ significantly in either experiment. Increased proliferation activity was detected in the presence of CD serum or after treatment with L-NAME. Cultures showed tube-like formations after 24 h treatment with CD serum.IBD sera evoked changes in the ratio of eNOS/iNOS, whereas did not influence the viability of HUVEC. These involved down-regulation of eNOS and up-regulation of iNOS simultaneously, leading to increased proliferation activity and possibly a reduced anti-inflammatory protection of endothelial cells.