Cannabinoid-mediated modulation of neuropathic pain and microglial accumulation in a model of murine type I diabetic peripheral neuropathic pain

Background

Despite the frequency of diabetes mellitus and its relationship to diabetic peripheral neuropathy (DPN) and neuropathic pain (NeP), our understanding of underlying mechanisms leading to chronic pain in diabetes remains poor. Recent evidence has demonstated a prominent role of microglial cells in neuropathic pain states. One potential therapeutic option gaining clinical acceptance is the cannabinoids, for which cannabinoid receptors (CB) are expressed on neurons and microglia. We studied the accumulation and activation of spinal and thalamic microglia in streptozotocin (STZ)-diabetic CD1 mice and the impact of cannabinoid receptor agonism/antagonism during the development of a chronic NeP state. We provided either intranasal or intraperitoneal cannabinoid agonists/antagonists at multiple doses both at the initiation of diabetes as well as after establishment of diabetes and its related NeP state.

Results

Tactile allodynia and thermal hypersensitivity were observed over 8 months in diabetic mice without intervention. Microglial density increases were seen in the dorsal spinal cord and in thalamic nuclei and were accompanied by elevation of phosphorylated p38 MAPK, a marker of microglial activation. When initiated coincidentally with diabetes, moderate-high doses of intranasal cannabidiol (cannaboid receptor 2 agonist) and intraperitoneal cannabidiol attenuated the development of an NeP state, even after their discontinuation and without modification of the diabetic state. Cannabidiol was also associated with restriction in elevation of microglial density in the dorsal spinal cord and elevation in phosphorylated p38 MAPK. When initiated in an established DPN NeP state, both CB1 and CB2 agonists demonstrated an antinociceptive effect until their discontinuation. There were no pronociceptive effects demonstated for either CB1 or CB2 antagonists.

Conclusions

The prevention of microglial accumulation and activation in the dorsal spinal cord was associated with limited development of a neuropathic pain state. Cannabinoids demonstrated antinociceptive effects in this mouse model of DPN. These results suggest that such interventions may also benefit humans with DPN, and their early introduction may also modify the development of the NeP state.     

Microbial community shifts influence patterns in tropical forest nitrogen fixation

The role of biodiversity in ecosystem function receives substantial attention, yet despite the diversity and functional relevance of microorganisms, relationships between microbial community structure and ecosystem processes remain largely unknown. We used tropical rain forest fertilization plots to directly compare the relative abundance, composition and diversity of free-living nitrogen (N)-fixer communities to in situ leaf litter N fixation rates. N fixation rates varied greatly within the landscape, and ‘hotspots’ of high N fixation activity were observed in both control and phosphorus (P)-fertilized plots. Compared with zones of average activity, the N fixation ‘hotspots’ in unfertilized plots were characterized by marked differences in N-fixer community composition and had substantially higher overall diversity. P additions increased the efficiency of N-fixer communities, resulting in elevated rates of fixation per nifH gene. Furthermore, P fertilization increased N fixation rates and N-fixer abundance, eliminated a highly novel group of N-fixers, and increased N-fixer diversity. Yet the relationships between diversity and function were not simple, and coupling rate measurements to indicators of community structure revealed a biological dynamism not apparent from process measurements alone. Taken together, these data suggest that the rain forest litter layer maintains high N fixation rates and unique N-fixing organisms and that, as observed in plant community ecology, structural shifts in N-fixing communities may partially explain significant differences in system-scale N fixation rates.     

Therapeutic Efficacy of Cintredekin Besudotox (IL13-PE38QQR) in Murine Lung Fibrosis Is Unaffected by Immunity to Pseudomonas aeruginosa Exotoxin A

Background

We have previously explored a therapeutic strategy for specifically targeting the profibrotic activity of IL-13 during experimental pulmonary fibrosis using a fusion protein comprised of human IL-13 and a mutated form of Pseudomonas aeruginosa exotoxin A (IL13-PE) and observed that the intranasal delivery of IL13-PE reduced bleomycin-induced pulmonary fibrosis through its elimination of IL-13-responsive cells in the lung. The aim of the present study was to determine whether the presence of an immune response to P. aeruginosa and/or its exotoxin A (PE) would diminish the anti-fibrotic properties of IL13-PE.

Methodology/Principal Findings

Fourteen days after P. aeruginosa infection, C57BL/6 mice were injected with bleomycin via the intratracheal route. Other groups of mice received 4 doses of saline or IL13-PE by either intranasal or intraperitoneal application, and were challenged i.t. with bleomycin 28 days later. At day 21 after bleomycin, all mice received either saline vehicle or IL13-PE by the intranasal route and histopatological analyses of whole lung samples were performed at day 28 after bleomycin. Intrapulmonary P. aeruginosa infection promoted a neutralizing IgG2A and IgA antibody response in BALF and serum. Surprisingly, histological analysis showed that a prior P. aeruginosa infection attenuated the development of bleomycin-induced pulmonary fibrosis, which was modestly further attenuated by the intranasal administration of IL13-PE. Although prior intranasal administration of IL13-PE failed to elicit an antibody response, the systemic administration of IL13-PE induced a strong neutralizing antibody response. However, the prior systemic sensitization of mice with IL13-PE did not inhibit the anti-fibrotic effect of IL13-PE in fibrotic mice.

Conclusions

Thus, IL13-PE therapy in pulmonary fibrosis works regardless of the presence of a humoral immune response to Pseudomonas exotoxin A. Interestingly, a prior infection with P. aeruginosa markedly attenuated the pulmonary fibrotic response suggesting that the immune elicitation by this pathogen exerts anti-fibrotic effects.     

Effect of active site and surface mutations on the reduction potential of yeast cytochrome c peroxidase and spectroscopic properties of the oxidized and reduced enzyme

The reduction potentials of 22 yeast cytochrome c peroxidase (CcP) mutants were determined at pH 7.0 in order to determine the effect of both heme pocket and surface mutations on the Fe(III)/Fe(II) redox couple of CcP, as well as to determine the range in redox potentials that could be obtained through point mutations in the enzyme. Spectroscopic properties of the Fe(III) and Fe(II) forms of the mutant enzymes are also reported. The mutations include variants in the distal and proximal heme pockets as well as on the enzyme surface and involve single, double, and triple point mutations. A spectrochemical redox titration technique used in this study gave an E(0') value of -189 mV for yeast CcP compared to a previously reported value of -194 mV determined by potentiometry [C.W. Conroy, P. Tyma, P.H. Daum, J.E. Erman, Biochim. Biophys. Acta 537 (1978) 62-69]. Both positive and negative shifts in the reduction potential from that of the wild-type enzyme were observed, spanning a range of 113 mV. The His-52-->Asn mutation gave the most negative potential, -259 mV, while a triple mutant in which the three distal pocket residues, Arg-48, Trp-51, and His-52, were all converted to leucine residues gave the most positive potential, -146 mV.     

Effects of nutritional restriction on nitrogen and carbon stable isotopes in growing seabirds

When using stable isotopes as dietary tracers it is essential to consider effects of nutritional state on isotopic fractionation. While starvation is known to induce enrichment of ¹⁵N in body tissues, effects of moderate food restriction on isotope signatures have rarely been tested. We conducted two experiments to investigate effects of a 50–55% reduction in food intake on δ¹⁵N and δ¹³C values in blood cells and whole blood of tufted puffin chicks, a species that exhibits a variety of adaptive responses to nutritional deficits. We found that blood from puffin chicks fed ad libitum became enriched in ¹⁵N and ¹³C compared to food-restricted chicks. Our results show that ¹⁵N enrichment is not always associated with food deprivation and argue effects of growth on diet–tissue fractionation of nitrogen stable isotopes (Δ¹⁵N) need to be considered in stable isotope studies. The decrease in δ¹³C of whole blood and blood cells in restricted birds is likely due to incorporation of carbon from ¹³C-depleted lipids into proteins. Effects of nutritional restriction on δ¹⁵N and δ¹³C values were relatively small in both experiments (δ¹⁵N: 0.77 and 0.41‰, δ¹³C: 0.20 and 0.25‰) compared to effects of ecological processes, indicating physiological effects do not preclude the use of carbon and nitrogen stable isotopes in studies of seabird ecology. Nevertheless, our results demonstrate that physiological processes affect nitrogen and carbon stable isotopes in growing birds and we caution isotope ecologists to consider these effects to avoid drawing spurious conclusions.     

The chemokine CCL6 promotes innate immunity via immune cell activation and recruitment

Septic syndrome is a consequence of innate immune failure. Recent studies showed that the CC chemokine CCL6 enhanced antimicrobial immunity during experimental sepsis through an unknown mechanism. The present study demonstrates that transgenic CCL6 expression abolishes mortality in a septic peritonitis model via the modulation of resident peritoneal cell activation and, more importantly, through the recruitment of IFN-producing NK cells and killer dendritic cells into the peritoneum. Thus, CCL6 attenuates the immune failure during sepsis, in part, through a protective type 1-cytokine mediated mechanism.     

Agmatine transport into spinal nerve terminals is modulated by polyamine analogs

Because alpha-synuclein (Snca) has a role in brain lipid metabolism, we determined the impact that Snca deletion had on whole brain lipid composition. We analysed masses of individual phospholipid (PL) classes and neutral lipid mass as well as PL acyl chain composition in brains from wild-type and Snca-/- mice. Although total brain PL mass was not altered, cardiolipin and phosphatidylglycerol mass decreased 16% and 27%, respectively, in Snca-/- mice. In addition, no changes were observed in plasmalogen or polyphosphoinositide mass. In ethanolamine glycerophospholipids and phosphatidylserine, docosahexaenoic acid (22 : 6n-3) was decreased 7%, while 16 : 0 was increased 1.1-fold and 1.4-fold, respectively. Surprisingly, brain cholesterol, cholesteryl ester, and triacylglycerol mass were increased 1.1-fold, 1.6-fold, and 1.4-fold, respectively in Snca-/- mice. In isolated myelin, cholesterol mass was also increased 1.3-fold, but because there was also a net increase in myelin PL mass, the cholesterol to PL ratio was unaltered. No changes in the expression of cholesterogenic enzymes were observed, suggesting these did not account for the observed changes in cholesterol. These data extend our previous results in astrocytes and kinetic studies in vivo demonstrating a role for Snca in brain lipid metabolism and demonstrate a clear impact on brain neutral lipid metabolism.     

Assay of the activity of malonyl-coenzyme A decarboxylase by gas chromatography-mass spectrometry

We developed a gas chromatography-mass spectrometry (GC-MS) assay to measure the activity of malonyl-coenzyme A (CoA) decarboxylase (MCD) in crude tissue homogenates. Liver extracts are incubated with [U-(13)C(3)]malonyl-CoA to form [U-(13)C(2)]acetyl-CoA by the action of MCD. The reaction mixture contains 2 mM ADP to prevent the hydrolysis of [1,2-(13)C(2)]acetyl-CoA by acetyl-CoA hydrolase present in the extracts. Newly formed [U-(13)C(2)]acetyl-CoA and internal standard of [(2)H(3),1-(13)C]acetyl-CoA are analyzed as thiophenol derivatives by GC-MS. This assay was applied to a study of the kinetics of MCD in rat liver. Using the Lineweaver-Burke plot of MCD kinetics, K(m) of 202microM and V(max) of 3.3micromol min(-1) (g liver)(-1) were calculated. The liver MCD activities (micromol min(-1) g(-1)+/-SD) in three groups of rats with different nutritional statuses-fed, 1-day fasted, and 2-day fasted-were 1.80+/-0.41, 2.59+/-0.37 (P<0.05), and 3.07+/-0.70 (P<0.05), respectively. We report a practical, nonradioactive, sensitive assay of MCD in crude tissue extract.     

Correlation of carboxylic acid pKa to protein binding and antibacterial activity of a novel class of bacterial translation inhibitors

Previously we reported the discovery and initial optimization of a novel anthranilic acid derived class of antibacterial agents which suffered from extensive protein binding. This report describes efforts directed toward understanding the relationship of the acidity of the carboxylic acid with the extent of protein binding. The pK(a) of the acid was modified via the synthesis of a number of anthranilic acid analogs which vary the aromatic ring substituent at the 4-position. The pK(a) and HSA binding constants have been determined for each of the analogs. Our results indicate a correlation between pK(a) and HSA K(d). The physical properties and antibacterial activities will be discussed as well as how these results help address the protein binding issue with this series of compounds.