Anders Kielland

AS04, an Aluminum Salt- and TLR4 Agonist-Based Adjuvant System, Induces a Transient Localized Innate Immune Response Leading to Enhanced Adaptive Immunity

Adjuvant System 04 (AS04) combines the TLR4 agonist MPL (3-O-desacyl-4′-monophosphoryl lipid A) and aluminum salt. It is a new generation TLR-based adjuvant licensed for use in human vaccines. One of these vaccines, the human papillomavirus (HPV) vaccine Cervarix, is used in this study to elucidate the mechanism of action of AS04 in human cells and in mice. The adjuvant activity of AS04 was found to be strictly dependent on AS04 and the HPV Ags being injected at the same i.m. site within 24 h of each other. During this period, AS04 transiently induced local NF-κB activity and cytokine production. This led to an increased number of activated Ag-loaded dendritic cells and monocytes in the lymph node draining the injection site, which further increased the activation of Ag-specific T cells. AS04 was also found to directly stimulate those APCs in vitro but not directly stimulate CD4+ T or B lymphocytes. These AS04-induced innate responses were primarily due to MPL. Aluminum salt appeared not to synergize with or inhibit MPL, but rather it prolonged the cytokine responses to MPL at the injection site. Altogether these results support a model in which the addition of MPL to aluminum salt enhances the vaccine response by rapidly triggering a local cytokine response leading to an optimal activation of APCs. The transient and confined nature of these responses provides further supporting evidence for the favorable safety profile of AS04 adjuvanted vaccines.

The effects of acute and chronic exercise on PGC‐1α, irisin and browning of subcutaneous adipose tissue in humans

Irisin was first identified as a peroxisome proliferator‐activated receptor γ co‐activator‐1α (PGC‐1α) dependent myokine with the potential to induce murine brown‐fat‐like development of white adipose tissue. In humans, the regulatory effect of training on muscle FNDC5mRNA expression and subsequently irisin levels in plasma is more controversial. We recruited 26 inactive men (13 normoglycaemic and normal weight, controls; and 13 slightly hyperglycaemic and overweight, pre‐diabetes group) aged 40–65 years for a 12‐week intervention of combined endurance and strength training with four sessions of training per week. Before and after the 12‐week intervention period, participants were exposed to an acute endurance workload of 45 min at 70% of VO2max, and muscle biopsies were taken prior to and after exercise. Skeletal muscle mRNA for PGC1A and FNDC5 correlated and both PGC1A and FNDC5mRNA levels increased after 12 weeks of training in both control and pre‐diabetes subjects. Circulating irisin was reduced in response to 12 weeks of training, and was increased acutely (~1.2‐fold) just after acute exercise. Plasma concentration of irisin was higher in pre‐diabetes subjects compared with controls. There was little effect of 12 weeks of training on selected browning genes in subcutaneous adipose tissue. UCP1mRNA did not correlate with FNDC5 expression in subcutaneous adipose tissue or skeletal muscle or with irisin levels in plasma. We observed no enhancing effect of long‐term training on circulating irisin levels, and little or no effect of training on browning of subcutaneous white adipose tissue in humans.

In vivo imaging of reactive oxygen and nitrogen species in inflammation using the luminescent probe L-012

Production of reactive oxygen and nitrogen species (ROS/RNS) is an important part of the inflammatory response, but prolonged elevated levels of ROS/RNS as under chronic inflammation can contribute to the development of disease. Monitoring ROS/RNS in living animals is challenging due to the rapid turnover of ROS/RNS and the limited sensitivity and specificity of ROS/RNS probes. We have explored the use of the chemiluminescent probe L-012 for noninvasive imaging of ROS/RNS production during inflammation in living mice. Various inflammatory conditions were induced, and L-012-dependent luminescence was recorded with an ultrasensitive CCD camera. Strong luminescent signals were observed from different regions of the body corresponding to inflammation. The signal was reduced by administration of the SOD mimetic tempol, the NADPH oxidase inhibitor apocynin, and the inhibitor of nitric oxide synthesis L-NAME, signifying the requirement for the presence of ROS/RNS. Additionally, the L-012 signal was abolished in mice with a mutation in the Ncf1 gene, encoding a protein in the NADPH oxidase complex 2, which generates ROS/RNS during inflammation. In conclusion, L-012 is well distributed in the mouse body and mediates a strong ROS/RNS-dependent luminescent signal in vivo and is useful for monitoring the development and regulation of inflammation in living organisms.

Synapsin Utilization Differs among Functional Classes of Synapses on Thalamocortical Cells

Several proteins in nerve terminals participate in synaptic transmission between neurons. The synapsins, which are synaptic vesicle-associated proteins, have widespread distribution in the brain and are assumed essential for sustained recruitment of vesicles during high rates of synaptic transmission. We compared the role of synapsins in two types of glutamatergic synapses on thalamocortical cells in the dorsal lateral geniculate nucleus of mice: retinogeniculate synapses, which transmit primary afferent input at high frequencies and show synaptic depression, and corticogeniculate synapses, which provide modulatory feedback at lower frequencies and show synaptic facilitation. We used electrophysiological methods to determine effects of gene knock-out of synapsin I and II on short-term synaptic plasticity in paired-pulse, pulse-train, and posttetanic potentiation paradigms. The gene inactivation changed the plasticity properties in corticogeniculate, but not in retinogeniculate, synapses. Immunostaining with antibodies against synapsins in wild-type mice demonstrated that neither synapsin I nor II occurred in retinogeniculate terminals, whereas both occurred in corticogeniculate terminals. In GABAergic terminals, only synapsin I occurred. In corticogeniculate terminals of knock-out mice, the density of synaptic vesicles was reduced because of increased terminal size rather than reduced number of vesicles and the intervesicle distance was increased compared with wild-type mice. In the retinogeniculate terminals, no significant morphometric differences occurred between knock-out and wild-type mice. Together, this indicates that synapsin I and II are not present in the retinogeniculate terminals and therefore are not essential for sustained, high-rate synaptic transmission.

Activity Patterns Govern Synapse-Specific AMPA Receptor Trafficking between Deliverable and Synaptic Pools

In single neurons, glutamatergic synapses receiving distinct afferent inputs may contain AMPA receptors (-Rs) with unique subunit compositions. However, the cellular mechanisms by which differential receptor transport achieves this synaptic diversity remain poorly understood. In lateral geniculate neurons, we show that retinogeniculate and corticogeniculate synapses have distinct AMPA-R subunit compositions. Under basal conditions at both synapses, GluR1-containing AMPA-Rs are transported from an anatomically defined reserve pool to a deliverable pool near the postsynaptic density (PSD), but further incorporate into the PSD or functional synaptic pool only at retinogeniculate synapses. Vision-dependent activity, stimulation mimicking retinal input, or activation of CaMKII or Ras signaling regulated forward GluR1 trafficking from the deliverable pool to the synaptic pool at both synapses, whereas Rap2 signals reverse GluR1 transport at retinogeniculate synapses. These findings suggest that synapse-specific AMPA-R delivery involves constitutive and activity-regulated transport steps between morphological pools, a mechanism that may extend to the site-specific delivery of other membrane protein complexes.

Molecular Nutrition Research—The Modern Way Of Performing Nutritional Science

In spite of amazing progress in food supply and nutritional science, and a striking increase in life expectancy of approximately 2.5 months per year in many countries during the previous 150 years, modern nutritional research has a great potential of still contributing to improved health for future generations, granted that the revolutions in molecular and systems technologies are applied to nutritional questions. Descriptive and mechanistic studies using state of the art epidemiology, food intake registration, genomics with single nucleotide polymorphisms (SNPs) and epigenomics, transcriptomics, proteomics, metabolomics, advanced biostatistics, imaging, calorimetry, cell biology, challenge tests (meals, exercise, etc.), and integration of all data by systems biology, will provide insight on a much higher level than today in a field we may name molecular nutrition research. To take advantage of all the new technologies scientists should develop international collaboration and gather data in large open access databases like the suggested Nutritional Phenotype database (dbNP). This collaboration will promote standardization of procedures (SOP), and provide a possibility to use collected data in future research projects. The ultimate goals of future nutritional research are to understand the detailed mechanisms of action for how nutrients/foods interact with the body and thereby enhance health and treat diet-related diseases.

Retinoid X receptor γ gene transcripts are expressed by a subset of early generated retinal cells and eventually restricted to photoreceptors

We have examined the distribution of the retinoid X receptor γ (RXRγ) in the developing chicken retina by using in situ hybridization and RNase protection assays. We detected RXRγ transcripts as early as 4 days of embryonic development (d4) in central regions of the retina, spreading to more peripheral regions by d8. The first few RXRγ‐positive cells were scattered within the depth of the retinal neuroepithelium, but as they increased in number they became localized predominantly to the apical (outer, ventricular) layer. The identity of the RXRγ‐positive cells at these stages is unknown, due to the lack of cell type‐specific markers. By d10, when photoreceptors and ganglion cells have been generated and begun to establish their definitive layers, RXRγ‐positive cells were virtually restricted to the photoreceptor layer, and maintained this distribution to posthatch stages. RNase protection assays were performed on whole retinae to verify the temporal pattern of in situ hybridization results and showed that between d5 and d16 there was a significant increase in the mRNA levels of the RXRγ2 isoform. Between d16 and early posthatch stages the level of RXRγ2 mRNA did not change significantly. Consistent with previous studies, mRNA levels of the RXRγ1 isoform were substantially lower than mRNA levels of the RXRγ2 isoform at all time points examined. These results demonstrate that RXRγ mRNA is expressed in photoreceptors in the developing chicken retina and implicate RXRγ as the earliest marker of photoreceptor differentiation documented to date. J. Comp. Neurol. 391:204–213, 1998.

Tracking Early Autoimmune Disease by Bioluminescent Imaging of NF-κB Activation Reveals Pathology in Multiple Organ Systems

It is desirable to have an early and sensitive detection marker of autoimmune disease in intact animals. Nuclear factor (NF)-kappaB is a transcription factor that is associated with inflammatory responses and immune disorders. Previously, we demonstrated that so-called idiotypic-driven T-B cell collaboration in mice doubly transgenic for paired immunoglobulin and T cell receptor transgenes resulted in a systemic autoimmune disease with systemic lupus erythematosus-like features. Here, we investigated NF-kappaB activation by including an NF-kappaB-responsive luciferase reporter transgene in this animal model. Triply transgenic mice developed bioluminescence signals from diseased organs before onset of clinical symptoms and autoantibody production, and light emissions correlated with disease progression. Signals were obtained from secondary lymphoid organs, inflamed intestines, skin lesions, and arthritic joints. Moreover, bioluminescence imaging and immunohistochemistry demonstrated that a minority of mice suffered from an autoimmune disease of the small intestine, in which light emissions correlated with antibodies against tissue transglutaminase and gliadin. Detection of luciferase by immunohistochemistry revealed NF-kappaB activation in collaborating B and T cells, as well as in macrophages. These results demonstrate that bioluminescent in vivo imaging of NF-kappaB activation can be used for early and sensitive detection of autoimmune disease in an experimental mouse model, offering new possibilities for the evaluation of anti-inflammatory drugs.

Regulation of angiopoietin‐like protein 4 production during and after exercise

Angiopoietin‐like protein 4 (ANGPTL4) may regulate lipoprotein lipase‐dependent plasma clearance of triacylglycerol from skeletal muscle during exercise. The aim of this study was to examine the importance of muscle in regulating ANGPTL4 in response to exercise. We sampled muscle biopsies and serum before, immediately after, and 2 h after 45 min of ergometer cycling. Sampling was done before and after a 12‐week training intervention in controls and dysglycemic subjects. Moreover, fat biopsies were taken before and after the training intervention. The regulation of ANGPTL4 was also investigated in several tissues of exercising mice, and in cultured myotubes. ANGPTL4 levels in serum and expression in muscle were highest 2 h after exercise in both groups. Whereas ANGPTL4 was higher in muscle of exercising controls as compared to dysglycemic subjects, the opposite was observed in serum. In exercising mice, Angptl4 mRNA showed both higher basal expression and induction in liver compared to muscle. Angptl4 mRNA was much higher in adipose tissue than muscle and was also induced by exercise. We observed two mRNA isoforms of ANGPTL4 in muscle and fat in humans. Both were induced by exercise in muscle; one isoform was expressed 5‐ to 10‐fold higher than the other. Studies in mice and cultured myotubes showed that both fatty acids and cortisol have the potential to increase ANGPTL4 expression in muscle during exercise. In conclusion, ANGPTL4 is markedly induced in muscle in response to exercise. However, liver and adipose tissue may contribute more than muscle to the exercise‐induced increase in circulating ANGPTL4.

AMPA and NMDA currents show different short‐term depression in the dorsal lateral geniculate nucleus of the rat

Paired‐pulse depression was studied at the glutamatergic synapse between retinal afferents and thalamocortical cells in the rat dorsal lateral geniculate nucleus. The main objective of this study was to examine the contributions of the pre‐ and postsynaptic sites to this depression by comparing AMPA‐ and NMDA‐receptor‐mediated responses. Equal depression of the two receptor components would indicate involvement of presynaptic mechanisms, while differences in depression would indicate involvement of postsynaptic mechanisms. Pharmacologically isolated AMPA‐ and NMDA‐receptor‐mediated currents were recorded using the whole‐cell patch‐clamp technique in acute thalamic slices. Both the AMPA and the NMDA components showed pronounced depression when retinal afferents were activated by paired pulses. The depression decayed within 5 s. The AMPA component was more strongly depressed than the NMDA component at paired‐pulse intervals ranging from 20 to 200 ms, suggesting the involvement of postsynaptic mechanisms. For intervals of 500 ms and longer, the depression of the two components was identical, suggesting the involvement of purely presynaptic mechanisms. The degree of depression measured without the use of pharmacological tools produced similar results, thus excluding the involvement of presynaptic ionotropic glutamate receptors. Cyclothiazide, a blocker of AMPA‐receptor desensitisation, reduced the difference in depression between the two components, suggesting that desensitisation of the AMPA receptors is a postsynaptic mechanism that contributes to the difference in depression between the AMPA and the NMDA components.