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Protease expression in experimental colitis
Extracellular matrix (ECM) is degraded by matrix metalloproteinases, collagenase, stromelysin and gelatinase, whose activity is strictly controlled by tissue inhibitor of metalloproteinase (TIMP). Excessive enzyme activity could lead to tissue destruction in inflammatory bowel disease (IBD). Using a rabbit model of chronic colitis we investigated the temporal and spatial distribution of these enzymes by immunolocalisation. 72 kD intracellular gelatinase was observed 3 h after initiation of colitis. At 6 h and 12 h, collagenase and, to a lesser extent, 72 kD and 95 kD gelatinase and stromelysin were all observed on the ECM in regions of mucosal ulceration. TIMP, however, was absent at these earlier times suggesting uncontrolled degradation of ECM, but by 24 h, it was expressed in mucosa adjacent to areas of ulceration. At 72 h and after one week, expression of collagenase declined and from two weeks until the ulcers resolved, stromelysin and gelatinase were found at the junction of normal and ulcerated tissue. TIMP expression remained constant until ulceration had healed at 6 weeks. In colon from animals killed at 0 h, no enzyme or TIMP expression was observed. Collagenase appears to be associated with the acute phase of ulcer formation, whereas stromelysin and gelatinase are predominant during healing. © 1994 Birkhäuser Verlag.
Metabolomics reveals distinct, antibody-independent, molecular signatures of MS, AQP4-antibody and MOG-antibody disease.
The overlapping clinical features of relapsing remitting multiple sclerosis (RRMS), aquaporin-4 (AQP4)-antibody (Ab) neuromyelitis optica spectrum disorder (NMOSD), and myelin oligodendrocyte glycoprotein (MOG)-Ab disease mean that detection of disease specific serum antibodies is the gold standard in diagnostics. However, antibody levels are not prognostic and may become undetectable after treatment or during remission. Therefore, there is still a need to discover antibody-independent biomarkers. We sought to discover whether plasma metabolic profiling could provide biomarkers of these three diseases and explore if the metabolic differences are independent of antibody titre. Plasma samples from 108 patients (34 RRMS, 54 AQP4-Ab NMOSD, and 20 MOG-Ab disease) were analysed by nuclear magnetic resonance spectroscopy followed by lipoprotein profiling. Orthogonal partial-least squares discriminatory analysis (OPLS-DA) was used to identify significant differences in the plasma metabolite concentrations and produce models (mathematical algorithms) capable of identifying these diseases. In all instances, the models were highly discriminatory, with a distinct metabolite pattern identified for each disease. In addition, OPLS-DA identified AQP4-Ab NMOSD patient samples with low/undetectable antibody levels with an accuracy of 92%. The AQP4-Ab NMOSD metabolic profile was characterised by decreased levels of scyllo-inositol and small high density lipoprotein particles along with an increase in large low density lipoprotein particles relative to both RRMS and MOG-Ab disease. RRMS plasma exhibited increased histidine and glucose, along with decreased lactate, alanine, and large high density lipoproteins while MOG-Ab disease plasma was defined by increases in formate and leucine coupled with decreased myo-inositol. Despite overlap in clinical measures in these three diseases, the distinct plasma metabolic patterns support their distinct serological profiles and confirm that these conditions are indeed different at a molecular level. The metabolites identified provide a molecular signature of each condition which is independent of antibody titre and EDSS, with potential use for disease monitoring and diagnosis.
Increased cortical neuronal responses to NMDA and improved attentional set-shifting performance in rats following prebiotic (B-GOS®) ingestion.
We have previously shown that prebiotics (dietary fibres that augment the growth of indigenous beneficial gut bacteria) such as Bimuno™ galacto-oligosaccharides (B-GOS®), increased N-methyl-D-aspartate (NMDA) receptor levels in the rat brain. The current investigation examined the functional correlates of these changes in B-GOS®-fed rats by measuring cortical neuronal responses to NMDA using in vivo NMDA micro-iontophoresis electrophysiology, and performance in the attentional set-shifting task. Adult male rats were supplemented with B-GOS® in the drinking water 3 weeks prior to in vivo iontophoresis or behavioural testing. Cortical neuronal responses to NMDA iontophoresis, were greater (+30%) in B-GOS® administered rats compared to non-supplemented controls. The intake of B-GOS® also partially hindered the reduction of NMDA responses by the glycine site antagonist, HA-966. In the attentional set-shifting task, B-GOS® -fed rats shifted from an intra-dimensional to an extra-dimensional set in fewer trials than controls, thereby indicating greater cognitive flexibility. An initial exploration into the mechanisms revealed that rats ingesting B-GOS® had increased levels of plasma acetate, and cortical GluN2B subunits and Acetyl Co-A Carboxylase mRNA. These changes were also observed in rats fed daily for 3 weeks with glyceryl triacetate, though unlike B-GOS®, cortical histone deacetylase (HDAC1, HDAC2) mRNAs were also increased which suggested an additional epigenetic action of direct acetate supplementation. Our data demonstrate that a pro-cognitive effect of B-GOS® intake in rats is associated with an increase in cortical NMDA receptor function, but the role of circulating acetate derived from gut bacterial fermentation of this prebiotic requires further investigation.
Exacerbation of Acute Traumatic Brain Injury by Circulating Extracellular Vesicles.
Inflammatory lesions in the brain activate a systemic acute-phase response (APR), which is dependent on the release of extracellular vesicles (EVs) into the circulation. The resulting APR is responsible for regulating leukocyte mobilization and subsequent recruitment to the brain. Factors that either exacerbate or inhibit the APR will also exacerbate or inhibit central nervous system (CNS) inflammation as a consequence and have the potential to influence ongoing secondary damage. Here, we were interested to discover how the circulating EV population changes after traumatic brain injury (TBI) and how manipulation of the circulating EV pool impacts on the outcome of TBI. We found the number of circulating EVs increased rapidly post-TBI, and this was accompanied by an increase in CNS and hepatic leukocyte recruitment. In an adoptive transfer study, we then evaluated the outcomes of TBI after administering EVs derived from either in vitro macrophage or endothelial cell lines stimulated with lipopolysaccharide (LPS), or from murine plasma from an LPS challenge using the air-pouch model. By manipulating the circulating EV population, we were able to demonstrate that each population of transferred EVs increased the APR. However, the characteristics of the response were dependent on the nature of the EVs; specifically, it was significantly increased when animals were challenged with macrophage-derived EVs, suggesting that the cellular origins of EVs may determine their function. Selectively targeting EVs from macrophage/monocyte populations is likely to be of value in reducing the impact of the systemic inflammatory response on the outcome of traumatic CNS injury.
Hepatic acute phase response protects the brain from focal inflammation during postnatal window of susceptibility.
Perinatal inflammation is known to contribute to neurodevelopmental diseases. Animal models of perinatal inflammation have revealed that the inflammatory response within the brain is age dependent, but the regulators of this variation remain unclear. In the adult, the peripheral acute phase response (APR) is known to be pivotal in the downstream recruitment of leukocytes to the injured brain. The relationship between perinatal brain injury and the APR has not been established. Here, we generated focal inflammation in the brain using interleukin (IL)-1β at postnatal day (P)7, P14, P21 and P56 and studied both the central nervous system (CNS) and hepatic inflammatory responses at 4 h. We found that there is a significant window of susceptibility in mice at P14, when compared to mice at P7, P21 and P56. This was reflected in increased neutrophil recruitment to the CNS, as well as an increase in blood-brain barrier permeability. To investigate phenomena underlying this window of susceptibility, we performed a dose response of IL-1β. Whilst induction of endogenous IL-1β or intercellular adhesion molecule (ICAM)-1 in the brain and induction of a hepatic APR were dose dependent, the recruitment of neutrophils and associated blood-brain barrier breakdown was inversely proportional. Furthermore, in contrast to adult animals, an additional peripheral challenge (intravenous IL-1β) reduced the degree of CNS inflammation, rather than exacerbating it. Together these results suggest a unique window of susceptibility to CNS injury, meaning that suppressing systemic inflammation after brain injury may exacerbate the damage caused, in an age-dependent manner.
Prebiotic attenuation of olanzapine-induced weight gain in rats: analysis of central and peripheral biomarkers and gut microbiota.
Olanzapine is an effective antipsychotic drug but since it causes significant weight gain, it is not well tolerated by psychosis patients. The prebiotic, B-GOS®, attenuates metabolic dysfunction in obese subjects, and in rodents, alters central NMDA receptors and may affect serotonin receptors that are relevant in psychosis. We have determined whether B-GOS® influenced olanzapine-associated weight gain and central NMDA and serotonin receptors. Circulating acetate, IL-1β, IL-8 and TNFα, liver acetyl-CoA carboxylase (ACC), white adipose tissue (WAT) acetate receptor GPR43, and specific faecal bacteria genera were also measured to provide mechanistic information. Adult female Sprague-Dawley rats were administered a B-GOS® (0.5 g/kg/day) solution or water for 21 days, and received a single, daily, intraperitoneal injection of olanzapine or saline on days 8-21. The intake of B-GOS® significantly attenuated olanzapine-induced weight gain without altering frontal cortex 5-HT2AR blockade. Cortical GluN1 levels were elevated by olanzapine in the presence of B-GOS®. Plasma acetate concentrations increased following B-GOS® or olanzapine administration alone, but reduced when prebiotic and drug were administered in combination. This pattern was paralleled by hepatic ACC mRNA expression. The abundance of WAT GPR43 mRNA was reduced by olanzapine, only in the absence of B-GOS®. Co-administration of B-GOS® and olanzapine also elevated plasma TNFα, which is reported to influence lipid metabolism. Finally, B-GOS® elevated faecal Bifidobacterium spp. and reduced some bacteria in the Firmicutes phylum, whilst olanzapine treatment either alone or with B-GOS®, was without effect. These data suggest that inclusion of B-GOS® as an adjunct to olanzapine treatment in schizophrenia may prevent weight gain and have benefits on cognitive function in psychosis. The role of acetate in these effects requires further investigation.
Multiparametric MR assessment of pediatric brain tumors.
MR assessment of pediatric brain tumors has expanded to include physiologic information related to cellular metabolites, hemodynamic and diffusion parameters. The purpose of this study was to investigate the relationship between MR and proton MR spectroscopic imaging in children with primary brain tumors. Twenty-one patients (mean age 9 years) with histologically verified brain tumors underwent conventional MR imaging, hemodynamic MR imaging (HMRI) and proton MR spectroscopic imaging (MRSI). Fourteen patients also had diffusion-weighted MR imaging (DWMRI). Metabolic indices including choline-containing compounds (Cho), total creatine (tCr) and lipids/lactate (L) were derived by proton MRSI, relative cerebral blood volume (rCBV) by HMRI, and apparent tissue water diffusion coefficients (ADC) by DWMRI. Variables were examined by linear regression and correlation as well as by ANOVA. Cho (suggestive of tumor cellularity and proliferative activity) correlated positively with rCBV, while the relationship between Cho and ADC (suggestive of cellular density) was inverse ( P<0.001). The relationship between rCBV and ADC was also inverse ( P=0.004). Cho and lipids (suggestive of necrosis and/or apoptosis) were not significantly correlated ( P=0.51). A positive relationship was found between lipids and ADC ( P=0.002). The relationships between Cho, rCBV, ADC and lipids signify that tumor physiology is influenced by the tumor's physical and chemical environment. Normalized Cho and lipids distinguished high-grade from low-grade tumors ( P<0.05). Multiparametric MR imaging using MRSI, HMRI and DWMRI enhances assessment of brain tumors in children and improves our understanding of tumor physiology while promising to distinguish higher- from lower-malignancy tumors, a distinction that is particularly clinically important among inoperable tumors.
Differential matrix metalloproteinase expression in cases of multiple sclerosis and stroke
D.C. Anthony, B. Ferguson, M.K. Matyzak, K.M. Miller, M.M. Esiri and V.H. Perry (1997) Neuropathology and Applied Neurobiology23, 406–415Differential matrix metalloproteinase expression in cases of multiple sclerosis and strokeMultiple sclerosis (MS) and stroke pathology are characterized by blood–brain barrier breakdown, leucocyte emigration, and tissue destruction. Each process is thought to involve the matrix metalloproteinases (MMP), but little is known of their expression. We undertook to investigate whether MMP expression is dependent on the nature of the CNS lesion and whether expression would coincide with the histopathology. MS or cerebral‐infarct tissue was examined for the presence of gelatinase‐A, gelatinase‐B, matrilysin and stromelysin‐1. Gelatinases A and B and matrilysin expression was found to be up‐regulated in microglia/macrophages within acute MS lesions. In active‐chronic MS lesions, matrilysin and gelatinase‐A expression was pronounced in the active borders. In chronic MS lesions, the expression of matrilysin was confined to macrophages within perivascular cuffs. The pattern of MMP expression in infarct lesions differed considerably. Gelatinase‐B was strongly expressed by neutrophils in tissue from patients up to 1 week after an infarct, whereas gelatinase‐A and matrilysin staining was much less marked. From 1 week to 5 years, neutrophils were absent and the large number of macrophages present were expressing matrilysin and gelatinase A. Only a low level of gelatinase‐A and matrilysin expression was observed in normal brain controls. Thus, MMPs are expressed in inflammatory lesions in the CNS, but their individual expression is dependent on the nature and chronicity of the lesion. However, the general pattern of expression, in perivascular cuffs and in active lesions, supports a role for these enzymes as mediators of blood–brain barrier breakdown and tissue destruction, both in MS and in cerebral ischaemia.