Influence of spinal cord transection on the presence and axonal transport of CGRP-, chromogranin A-, VIP-, synapsin I-, and synaptophysin-like immunoreactivities in rat motor nerve

Using immunofluorescence and cytofluorimetric scanning (CFS), we investigated the short-term (1-7 days) influence of lower thoracic spinal cord transection on lumbar motor neurons. The content of calcitonin gene-related peptide- (CGRP) like immunoreactivity (LI), chromogranin A (Chr A) -LI, vasoactive intestinal polypeptide (VIP)-LI, Syn I-LI, and synaptophysin (p38)-LI in motor perikarya, and the anterograde and retrograde axonal transport of these substances in the sciatic nerve, were studied in nerve crush (6 h) experiments. During the week after transection, CGRP-LI in perikarya decreased, whereas Chr A-LI increased. VIP-LI, co-localized with Chr A-LI in motor perikarya, did not change after transection. The antero- and retrograde transport of CGRP-LI in the sciatic nerve, occurring in both motor and sensory axons, appeared unchanged in cytofluorimetric scanning (CFS) graphs, but the microscopical picture clearly showed that large motor axons had a decreased content of CGRP-LI at 3 and 7 days posttransection, whereas thinner axons were unchanged in fluorescence intensity. The anterograde transport of Chr A-LI, present in both motor and postganglionic adrenergic axons, was decreased 1 and 3 days after lesion, but returned to control by day 7. There was a marked decrease in anterograde transport of VIP-LI, present mainly in postganglionic sympathetic axons, at day 3, but at 7 days transport was normal. The amounts of transported p38, the synaptic vesicle marker, were in the normal range during the whole period. Syn I-LI accumulation anterogradely was somewhat decreased at 3 and 7 days posttransection, and at 1 day the retrograde accumulation was significantly increased. The results suggest that removal of supraspinal input to intact lower motor neurons causes alterations in metabolism and axonal transport of organelle-associated substances, partly probably related to the complex pattern of transmitter leakage from degenerating, descending nerve terminals. These alterations appear to take place also in postganglionic sympathetic neurons in the sciatic nerve, that originate in the lumbar sympathetic chain. © 1992 John Wiley & Sons, Inc.     

Protein conformational exchange measured by 1H R1ρ relaxation dispersion of methyl groups

Activated dynamics plays a central role in protein function, where transitions between distinct conformations often underlie the switching between active and inactive states. The characteristic time scales of these transitions typically fall in the microsecond to millisecond range, which is amenable to investigations by NMR relaxation dispersion experiments. Processes at the faster end of this range are more challenging to study, because higher RF field strengths are required to achieve refocusing of the exchanging magnetization. Here we describe a rotating-frame relaxation dispersion experiment for ¹H spins in methyl ¹³CHD₂ groups, which improves the characterization of fast exchange processes. The influence of ¹H–¹H rotating-frame nuclear Overhauser effects (ROE) is shown to be negligible, based on a comparison of R _1ρ relaxation data acquired with tilt angles of 90° and 35°, in which the ROE is maximal and minimal, respectively, and on samples containing different ¹H densities surrounding the monitored methyl groups. The method was applied to ubiquitin and the apo form of calmodulin. We find that ubiquitin does not exhibit any ¹H relaxation dispersion of its methyl groups at 10 or 25 °C. By contrast, calmodulin shows significant conformational exchange of the methionine methyl groups in its C-terminal domain, as previously demonstrated by ¹H and ¹³C CPMG experiments. The present R _1ρ experiment extends the relaxation dispersion profile towards higher refocusing frequencies, which improves the definition of the exchange correlation time, compared to previous results.     

Effect of maternal feed restriction during pregnancy on glucose tolerance in the adult guinea pig

Maternal nutrient restriction and impaired fetal growth are associated with postnatal insulin resistance, hyperinsulinemia, and glucose intolerance in humans but not consistently in other species, such as the rat or sheep. We therefore determined the effect of mild (85% ad libitum intake/kg body wt) or moderate (70% ad libitum intake/kg body wt) maternal feed restriction throughout pregnancy on glucose and insulin responses to an intravenous glucose tolerance test (IVGTT) in the young adult guinea pig. Maternal feed restriction reduced birth weight (mild and moderate: both P < 0.02) in male offspring. Moderate restriction increased plasma glucose area under the curve (P < 0.04) and decreased the glucose tolerance index (K(G)) (P < 0.02) during the IVGTT in male offspring compared with those of mildly restricted but not of ad libitum-fed mothers. Moderate restriction increased fasting plasma insulin (P < 0.04, adjusted for litter size) and the insulin response to IVGTT (P < 0.001), and both moderate and mild restriction increased the insulin-to-glucose ratio during the IVGTT (P < 0.003 and P < 0.02) in male offspring. When offspring were classed into tertiles according to birth weight, glucose tolerance was not altered, but fasting insulin concentrations were increased in low compared with medium birth weight males (P < 0.03). The insulin-to-glucose ratio throughout the IVGTT was increased in low compared with medium (P < 0.01) or high (P < 0.05) birth weight males. Thus maternal feed restriction in the guinea pig restricts fetal growth and causes hyperinsulinemia in young adult male offspring, suggestive of insulin resistance. These findings suggest that mild to moderate prenatal perturbation programs postnatal glucose homeostasis adversely in the guinea pig, as in the human.     

Optimizing immobilization on two-dimensional carboxyl surface: pH dependence of antibody orientation and antigen binding capacity

The performance of immunosensors is highly dependent on the amount of immobilized antibodies and their remaining antigen binding capacity. In this work, a method for immobilization of antibodies on a two-dimensional carboxyl surface has been optimized using quartz crystal microbalance biosensors. We show that successful immobilization is highly dependent on surface pK_a, antibody pI, and pH of immobilization buffer. By the use of EDC/sulfo-NHS (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysulfosuccinimide) activation reagents, the effect of the intrinsic surface pK_a is avoided and immobilization at very low pH is therefore possible, and this is important for immobilization of acidic proteins. Antigen binding capacity as a function of immobilization pH was studied. In most cases, the antigen binding capacity followed the immobilization response. However, the antigen-to-antibody binding ratio differed between the antibodies investigated, and for one of the antibodies the antigen binding capacity was significantly lower than expected from immobilization in a certain pH range. Tests with anti-Fc and anti-Fab₂ antibodies on different antibody surfaces indicated that the orientation of the antibodies on the surface had a profound effect on the antigen binding capacity of the immobilized antibodies.     

Estrogen regulates T helper 17 phenotype and localization in experimental autoimmune arthritis

IntroductionThe incidence and progression of many autoimmune diseases are sex-biased, which might be explained by the immunomodulating properties of endocrine hormones. Treatment with estradiol potently inhibits experimental autoimmune arthritis. Interleukin-17-producing T helper cells (Th17) are key players in several autoimmune diseases, particularly in rheumatoid arthritis. The aim of this study was to investigate the effects of estrogen on Th17 cells in experimental arthritis.MethodsOvariectomized DBA/1 mice treated with 17β-estradiol (E2) or placebo were subjected to collagen-induced arthritis (CIA), and arthritis development was assessed. Th17 cells in joints and lymph nodes were studied by flow cytometry. Lymph node Th17 cells were also examined in ovariectomized estrogen receptor α–knockout mice (ERα^−/−) and wild-type littermates, treated with E2 or placebo and subjected to antigen-induced arthritis.ResultsE2-treated mice with established CIA showed reduced severity of arthritis and fewer Th17 cells in joints compared with controls. Interestingly, E2-treated mice displayed increased Th17 cells in lymph nodes during the early phase of the disease, dependent on ERα. E2 increased the expression of C-C chemokine receptor 6 (CCR6) on lymph node Th17 cells as well as the expression of the corresponding C-C chemokine ligand 20 (CCL20) within lymph nodes.ConclusionsThis is the first study in which the effects of E2 on Th17 cells have been characterized in experimental autoimmune arthritis. We report that E2 treatment results in an increase of Th17 cells in lymph nodes during the early phase of arthritis development, but leads to a decrease of Th17 in joints during established arthritis. Our data suggest that this may be caused by interference with the CCR6-CCL20 pathway, which is important for Th17 cell migration. This study contributes to the understanding of the role of estrogen in the development of autoimmune arthritis and opens up new fields for research concerning the sex bias in autoimmune disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0548-y) contains supplementary material, which is available to authorized users.     

Microsatellites in the Sand Lizard (Lacerta agilis): Description, Variation, Inheritance, and Applicability

We developed microsatellite markers for the sand lizard (Lacerta agilis) to enable investigations of the genetic variability within and among populations with a heterogeneous spatial distribution in Sweden. The populations, which could not be characterized by variation in allozymes or mitochondrial DNA, had a substantial level of variability in microsatellite loci. However, the variability in Swedish populations was limited compared to a large, outbred Hungarian population. In the sand lizard, the number of (GT/CA) _n repeats was approximately three times higher than that for (CT/GA) _n. The number of repeats and the frequency of microsatellites were within the range reported for other species. Three of nine microsatellite loci showed alleles that could not be amplified, which is in agreement with recent reports describing microsatellite “null alleles” as a common occurrence. We discuss the caution which this calls for when calculating paternity probabilities and when estimating between-population allelic differentiation. A potential problem with different mutation rates for alleles within the same locus is discussed.     

Anti-dsDNA Antibodies Promote Initiation,and Acquired Loss of Renal Dnase1 Promotes Progression of Lupus Nephritis in Autoimmune (NZBxNZW)F1 Mice

Background

Lupus nephritis is characterized by deposition of chromatin fragment-IgG complexes in the mesangial matrix and glomerular basement membranes (GBM). The latter defines end-stage disease.

Methodology/Principals

In the present study we determined the impact of antibodies to dsDNA, renal Dnase1 and matrix metalloprotease (MMP) mRNA levels and enzyme activities on early and late events in murine lupus nephritis. The major focus was to analyse if these factors were interrelated, and if changes in their expression explain basic processes accounting for lupus nephritis.

Findings

Early phases of nephritis were associated with chromatin-IgG complex deposition in the mesangial matrix. A striking observation was that this event correlated with appearance of anti-dsDNA antibodies and mild or clinically silent nephritis. These events preceded down-regulation of renal Dnase1. Later, renal Dnase1 mRNA level and enzyme activity were reduced, while MMP2 mRNA level and enzyme activity increased. Reduced levels of renal Dnase1 were associated in time with deficient fragmentation of chromatin from dead cells. Large fragments were retained and accumulated in GBM. Also, since chromatin fragments are prone to stimulate Toll-like receptors in e.g. dendritic cells, this may in fact explain increased expression of MMPs.

Significance

These scenarios may explain the basis for deposition of chromatin-IgG complexes in glomeruli in early and late stages of nephritis, loss of glomerular integrity and finally renal failure.