Targeting the Fas/FasL system in Rheumatoid Arthritis therapy: Promising or risky?

Rheumatoid Arthritis (RA) is a chronic inflammatory disease affecting synovial joints. Tumor necrosis factor (TNF) α is a key component of RA pathogenesis and blocking this cytokine is the most common strategy to treat the disease. Though TNFα blockers are very efficient, one third of the RA patients are unresponsive or present side effects. Therefore, the development of novel therapeutic approaches is required. RA pathogenesis is characterized by the hyperplasia of the synovium, closely associated to the pseudo-tumoral expansion of fibroblast-like synoviocytes (FLS), which invade and destroy the joint structure. Hence, depletion of RA FLS has been proposed as an alternative therapeutic strategy. The TNF family member Fas ligand (FasL) was reported to trigger apoptosis in FLS of arthritic joints by binding to its receptor Fas and therefore suggested as a promising candidate for targeting the hyperplastic synovial tissue. However, this cytokine is pleiotropic and recent data from the literature indicate that Fas activation might have a disease-promoting role in RA by promoting cell proliferation. Therefore, a FasL-based therapy for RA requires careful evaluation before being applied. In this review we aim to overview what is known about the apoptotic and non-apoptotic effects of Fas/FasL system and discuss its relevance in RA.     

Endocytosis of G Protein-Coupled Receptors and Their Ligands: Is There a Role in Metal Trafficking?

The prevalence of metal dysregulation in many neurodegenerative and neurocognitive disorders has compelled many studying such diseases to investigate the mechanisms underlying metal regulation in the central nervous system. Metal homoeostasis is often complex, with sophisticated, multilayered pathways in operation. G protein-coupled receptors are omnipresent on cell membranes and have intriguing mechanisms of endocytosis and trafficking that may be useful in metal homoeostasis. Indeed, many receptors and/or their cognate ligands are able to bind metals, and in many cases metals are considered to have neuromodulatory roles as a result of receptor binding. In this mini-review, we outline the structural and functional aspects of G protein-coupled receptors with a focus on the mechanisms leading to endocytosis and cellular trafficking. We further highlight how this may help in the trafficking of metal ions, notably copper.     

Is Ultrasound a Better Target than Clinical Disease Activity Scores in Rheumatoid Arthritis with Fibromyalgia? A Case-Control Study

Objectives

Our goal is to study the correlations among gray-scale seven-joint ultrasound score (GS-US7), power Doppler seven-joint ultrasound score (PD-US7), disease activity score-28 joints (DAS28), simplified disease activity index (SDAI) and clinical disease activity index (CDAI) in patients with and without fibromyalgia (FM).

Methods

A matched case-control study included all patients consecutively seen in the Rheumatoid Arthritis (RA) Clinic. Participants were allocated into one of two groups: RA with FM and RA without FM. Ultrasound (US) and clinical scoring were blinded for the presence of FM. Medians and proportions were compared by Mann-Whitney’s test and McNemar’s test, respectively. Spearman’s rank correlation coefficients (r_s) were calculated among clinical and US scores and differences were tested by r-to-z transformation test.

Results

Seventy-two women were included, out of 247 RA patients, mostly white, with median (IQR) age of 57.5 (49.3–66.8) years, with RA symptoms for 13.0 (6.0–19.0) years and FM symptoms for 6.0 (2.0–15.0) years. Disease-modifying antirheumatic drugs, non-steroidal anti-inflammatory drugs and prednisone use was comparable between groups. Objective activity parameters were not different between groups. RA patients with FM had greater DAS28, SDAI and CDAI but similar GS-US7 and PD-US7. GS-US7 correlated with DAS28, SDAI and CDAI in patients with and without FM (r_s = 0.36–0.57), while PD-US7 correlated with clinical scores only in patients without FM (r_s = 0.35–0.38).

Conclusion

To our knowledge, this is the first study to demonstrate that ultrasound synovitis scores are not affected by FM in RA patients. PD-US7 performed better than GS-US7 in long-standing RA patients with DAS28, SDAI or CDAI allegedly overestimated due to FM. Since sonographic synovitis predicts erosion better than swollen joint count, C-reactive protein and erythrocyte sedimentation rate, US should be considered a promising treatment target in RA patients with FM.     

What Is the Role of ε in the Escherichia coli ATP Synthase?

The ATP synthase from Escherichia coli is a prototype of the ATP synthases that are found in many bacteria, in the mitochondria of eukaryotes, and in the chloroplasts of plants. It contains eight different types of subunits that have traditionally been divided into F₁, a water-soluble catalytic sector, and F_o, a membrane-bound ion transporting sector. In the current rotary model for ATP synthesis, the subunits can be divided into rotor and stator subunits. Several lines of evidence indicate that is one of the three rotor subunits, which rotate through 360 degrees. The three-dimensional structure of is known and its interactions with other subunits have been explored by several approaches. In light of recent work by our group and that of others, the role of in the ATP synthase from E. coli is discussed.     

Thermoregulation: What Role for UCPs in Mammals and Birds?

Mammals and birds are endotherms and respond to cold exposure by the means of regulatory thermogenesis, either shivering or non-shivering. In this latter case, waste of cell energy as heat can be achieved by uncoupling of mitochondrial respiration. Uncoupling proteins, which belong to the mitochondrial carrier family, are able to transport protons and thus may assume a thermogenic function. The mammalian UCP1 physiological function is now well understood and gives to the brown adipose tissue the capacity for heat generation. But is it really the case for its more recently discovered isoforms UCP2 and UCP3? Additionally, whereas more and more evidence suggests that non-shivering also exists in birds, is the avian UCP also involved in response to cold exposure? In this review, we consider the latest advances in the field of UCP biology and present putative functions for UCP1 homologues.     

Tau Aggregation in Alzheimer's Disease: What Role for Phosphorylation?

The crucial role of the neuronal Tau protein in microtubule stabilization and axonal transport suggests that too little or too much Tau might lead to neuronal dysfunction. The presence of a hyper phosphorylated but non aggregated molecule as a toxic species that might sequester normal Tau is discussed. We present recent in vitro results that might allow us to dissect the role of individual phosphorylation sites on its structure and function. We also discuss in this review the role of phosphorylation for the aggregation of the neuronal Tau protein, and compare it to the aggregation induced by external poly anions.Key Words: Tau, phosphorylation, paired helical filaments, microtubule, Alzheimer''s diseaseAlzheimer''s disease and its concomitant cognitive decline form a grim perspective for society, especially as the average life span of our population is expected to increase. This is even more the case as part of the basic knowledge concerning the disease is still under discussion. Indeed, contrary to other major disease classes such as cancer where a number of biological players have been well defined and have turned into potential targets for drug development, the molecular events leading to neuronal degeneration and ensuing cognitive decline are not completely understood. Even more, in a remarkable paradigm shift, both the extracellular β amyloid plaques and intracellular neuronal filaments and tangles, previously thought of as the main molecular markers and also the main culprits for the disease, are now considered as an ultimate rescue mechanism of the diseased brain.¹ This change in perception, equally found in other neuronal diseases such as Huntington''s disease,² deprives us from an obvious pharmacological target, even though it is not clear what we get in exchange. Alzheimer derived diffusible ligands (ADDLs) are β amyloid peptide oligomers,³ without a clear definition though of their precise molecular content and conformation. As for Tau, the major component of the neuronal filaments and tangles,⁴ a hyperphosphorylated but soluble form rather than the aggregated protein might be the toxic species. Finally, even the two opposing viewpoints of “gain of toxic function” versus “loss of physiological function” have not yet been sorted out for neither molecular marker, be it for β amyloid⁵ or Tau.⁶ The functional overlap between Tau and other microtubule associated proteins (MAPs), leading to the absence of a clear phenotype for Tau knockout mice, does not even lead to a clear cut answer to this question. However, both an overproduction of the longer β amyloid [1–42] peptide and an abnormal (hyper)phosphorylation of Tau seem related to the disease, and it remains a major challenge to tease out the precise role of these components in the disease progression.From a clinical perspective, Alzheimer''s diseased neurofibrillary pathology has been post mortem scored according to the Braak rules.⁷ This latter staging is based upon quantifying the neurofibrillary lesions in distinct regions from the brain using a silver iodate technique originally proposed by Gallyas.⁸ Technical constraints, however, have limited this staging to research centers, and have prompted the same group to develop a more routinely accessible immunochemical method.⁹ The latter uses the AT8 antibody, that immunostains a hyperphosphorylated Tau form, be it in its soluble or aggregated form.¹⁰ Importantly, the comparative imaging of brain slices with silver or with the antibody allow a nearly identical staging, establishing an unambiguous link between hyperphosphorylation and the presence of tangles. In this review, we want to focus on the link between (hyper)phosphorylation and disease related aspects of Tau, and want to discuss how in vitro studies might shed further light on the link between Tau post translational modifications, its aggregation and the general modulation of its functional aspects.A first question concerns a clear definition of “hyper ” and “abnormal” phosphorylation. Normal Tau contains 2–3 phosphate groups, assuring the dynamic character of the microtubule network (see below). Tau in its aggregated form (as Paired Helical Filaments (PHFs) or Straight Filaments (SF)) contains 5–9 moles of phosphate/ mole of the protein, defining it as hyper phosphorylated.¹¹ Overlap exists between the AD and normal adult patterns of phosphorylation, making the quantitative differences in the level of phosphate incorporation one of the decisive parameters.¹² Specific phosphorylation patterns also seem generated in the disease, and these form the basis for a large class of AD specific antibodies, including the above mentioned AT-8 antibody¹⁰ with specificity for both phospho-Ser199/phospho-Ser202 and phosphoThr205. All these data indicate that some phospho “bar code” might exist, whereby some sites are important for its physiological role of microtubule dynamics regulator, whereas another set (overlapping or not with the previous one) leads to aggregation into PHFs, degradation and/or toxic function.Untangling this code will be a major enterprise, largely due to the large number of phosphorylation sites on Tau together with the complex interplay of the different kinases involved. Underlying many of the difficulties is the analytical problem of characterizing samples at a qualitative (what sites?) and quantitative (what degree of phosphate incorporation?) level. Both mass spectrometry and immunochemistry have well recognized advantages of sensitivity for characterizing a phosphorylation pattern of Tau samples derived from in vivo material, and the recent demonstration of top down mass spectrometry for the characterization of complex protein molecules without previous digestion has the potential of opening up a novel observation window for such complex patterns.¹³ We recently have demonstrated that NMR spectroscopy equally might play a role in characterizing a complex phosphorylation pattern.¹⁴ Although plagued by an extremely low sensitivity compared to the above mentioned methods and requiring a stable isotope labelled substrate protein, it has the potential to answer both questions of what site(s) are modified and to what extent, and its non destructive character leads to well-characterized samples that then can be used for structural and functional assays. As an example, after a full characterization of the cAMP dependent kinase (PKA) generated phospho-pattern (Fig. 1A), we acrylodan labelled the same sample and used it to quantify the binding parameters to taxol-stabilized microtubules. This allowed us to demonstrate that phosphorylation of the Ser214 position causes an affinity drop by two orders of magnitude (Fig. 1B), without detaching this part of the protein from the microtubule surface. Despite this, the protein is not completely devoid of polymerization activity (Fig. 1c), underscoring the complexity of relationship between phosphorylation status and activity.Open in a separate windowFigure 1(Left) NMR assignment of the phosphorylation pattern of Tau after incubation with PKA¹⁴. (Right) Effect of a single phosphorylation event at Ser214 on the microtubule binding properties¹⁵ (right, top) or on its tubulin polymerizing capacity (right, bottom). In both panes, the upper curve is Tau, and the lower one pSer214 Tau. The affinity of acrylodan labeled Tau towards taxol stabilzed microtubules was measured by FRET for Tau (solid curve) or pSer214 Tau (dotted line), whereas turbidity was used to evaluate the polymerization of tubulin into microtubules.The axon of the mature neuron is characterized by a polarized microtubule orientation, while dendrites contain microtubules of mixed polarity.¹⁶ At the core of the complex neuronal transport machinery that assures correct subcellular localization of organelles, mRNAs and proteins, the dynamic stability of the microtubular network is of uttermost importance for the correct functioning of the neuron.¹⁷ Tau localizes mostly to axons, whereas MAP2 localization is largely restriced to the somatodendritic compartment. As long-distance trafficking uses mainly the axonal microtubule railway and Tau does (de)stabilize this network, it is of no surprise that a deregulation of its expression and/or phosphorylation level can lead to defects in axonal transport such as found in the early stages of AD¹⁸ or even at the later stages of the disease.¹⁹ Overexpression of the longest human tau isoform in wild-type mice leads to motor defects similar to those observed in progressive supranuclear palsy, another tauopathy,²⁰ but crossing these mice with constitutively active Gsk3β transgenic mice reduces importantly the number of axonal dilatations in brain and spinal cord, the axonal degeneration and muscular atrophy, and alleviates practically all motor problems.²¹ The amount of Tau associated with microtubules was reduced by 50% in preparations from brain and spinal cord of these mice that overexpress both human Tau and Gsk3β compared to the hTau transgenic mice. In vitro, Tau accumulation at the surface of taxol stabilized microtubules has been observed, albeit with a lower affinity than the direct interaction,²² and these aggregates might correspond to the traffick deregulating patches of Tau causing the axonopathy. In the hTau/Gsk3β mouse model, however, the authors did not detect true Tau filaments or neurofibrillary tangles, suggesting that the phosphorylation by this sole enzyme is enough to create a phosphorylation pattern of Tau that avoids its accumulation on the microtubule surface, but that does not lead to its aggregation into PHFs. Using a different mouse model overexpressing the naturally aggregating P301L hTau mutant, Le Corre et al. tested a small molecule inhibitor of the Erk2 kinase (with, however, a similar inhibition for other kinases such as cdc2, Gsk3β, PKA and PKC), and found that this compound does reduce motor impairments in a P301L Tau transgenic mouse model.²³ Without affecting tangle counts, the inhibitor causes a reduction of soluble aggregated hyperphosphorylated tau, although the exact nature of this species remains to be defined. The conclusions of this study with the kinase inhibitor build upon previous work by the group of Iqbal, who showed that (i) a hyperphosphorylated but soluble form of Tau (AD P-Tau) exists¹¹ (ii) it interacts with normal Tau,²⁴ and (iii) aggregation of this AD P-Tau into filaments neutralizes this interaction.²⁵ This species would not interact with tubulin, but even when present in a minor concentration, it would form a sink for the normal Tau, thereby leading to disruption of the microtubular network. When the disease progresses, the concentration of AD P-Tau increases, leading eventually to its aggregated form. Using an inducible model of the same hTau (P301L) transgenic mouse, Santa Cruz et al. showed that the soluble hyperphosphorylated species leads to neuronal degeneration, and this irrespective of ongoing tangle formation after the shutting off of the transgene.²⁶Whereas these studies tend to indicate that the filaments of aggregated Tau are not the main culprit, questions remain as for the identity of the species that would be responsible for the sequestration of normal Tau, and hence lead to microtubule impairment. In vitro studies hereby can play an important role, as they can hopefully reproduce and subsequently allow the identification of the molecular features that define these species. We have thus set out to identify the kinase(s) that might lead to a species that (i) interacts with normal Tau with such an affinity that it might disrupt the Tau:microtubule interaction, and (ii) that might lead to the formation of amyloid aggregates without the addition of any anionic cofactors. As for the first requirement, recent data by FRET spectroscopy using an acrylodan labelled Tau and taxol stabilized microtubules have shown that the affinity of Tau for the microtubular surface is high, with a dissociation constant K_D of the order of 20 nM.^15,27 When Tau is the polymerizing agent, the binding is even characterized by a quasi irreversible component.²⁷ Finally, the tubulin concentration in the neuronal axon is high, so for P-Tau to compete successfully for this MT associated Tau, one would need a very high affinity constant. Alternatively, two aspects of the same phosphorylation event(s) might reinforce this scenario, whereby a given subset of kinases generates the AD P-Tau species, and another one leads to destabilization of the Tau:MT interaction. In this aspect, it is interesting to note that the sole phosphorylation of Ser214 by PKA can lead to a hundred fold decrease in affinity for the MT surface (Fig. 1B).As for the molecular aspects of Tau aggregation, most if not all of the present work has followed up on the initial observation that the addition of poly anions such as heparin can promote the aggregation of Tau into PHFs that under the electron microscope have the same aspect as those fibers isolated form the brains of AD patients.²⁸ Because later on, other poly anions such as the surface of arachidonic acid micelles or RNA equally were found to promote the fibrilization process,^29,30 charge compensation rather than the precise anion seems to play an important role in the process. Following up on earlier mapping studies of the core region with proteases,³¹ we have recently NMR spectroscopy to (i) define the immobile core region with a per-residue precision³² and (ii) map the interaction between heparin fragments and Tau.³³ We confirmed the pronounced interaction of heparin with both the regions up and downstream of the microtubule binding repeats (MTBRs), but observed equally a strong interaction with the second and third repeat.³³ Of special interest was the observation that all heparin seems integrated in the fiber, as all visible signals of mobility retaining regions resonated at exactly the same frequency as the free Tau protein. EPR data on full length Tau PHF suggested a model for the fibers with a parallel in-register stacking of β strand,³⁴ similar to the recently obtained high resolution data on crystals³⁵ or fibers³⁶ derived from a prion peptide, or of fibers from β amyloid peptide.³⁷ Those peptide arrangements show a “dry” interface where the shape complementarity of facing side chains leaves little room even for water molecules,³⁵ further excluding the possibility that heparin intercalates between the facing β sheets. Considering that the NMR invisible solid like core region in the heparin induced PHFs spanning over hundred amino acids is not necessarily the true amyloid region, we can imagine that this region consists both of immobilized but disordered regions, and of genuine amyloid regions defined by some regular stacking. In other systems, this amyloid region consists of rather small peptides, and if we consider the R2 and R3 repeats, we can imagine that they would be no longer than the V₂₇₅QIINK₂₈₀ (in PHF6*) or V₃₀₆QIVYK₃₁₁ (in PHF6) hexapeptides, previously identified by peptide mapping studies as the aggregation nuclei.^38,39 Stacking of these peptides into parallel in register β sheets would however lead to the formation of ladder-like intermolecular stretches of the same residues. The K₂₈₀–K₂₈₁ and K₃₁₁P₃₁₂ motifs would create a continuous stretch of positive charges with accompanying strong electrostatic repulsion, unless a mechanism of charge neutralization is provided. One of such mechanisms is the deletion of at least one lysine, and ΔK₂₈₀ indeed is a mutant which aggregates more rapidly.⁴⁰ The in vitro study suggests a model where the heparin polymer wraps tightly around the outer surface of the (double) pleated sheets, and thereby neutralizes the inhibitory charge repulsions that would occur in a continuous but intermolecularly formed polylysine stretch. The heterogeneous nature of most heparin preparations thereby might lead to fibers of lesser regularity than those formed by isolated synthetic peptides. Solid state NMR and/or crystallography, as techniques that might resolve the recent controversy concerning the structure of the core region—only cross β strands^41,42 or equally some α helical structure^43,44—will require homogeneous preparations of filaments, and will have to distinguish between the amorphous and truly amyloid phases in this core. Our finding that subtle variations in size and/or charge distribution between two batches of the same commercial heparin can lead to fiber formation of all Tau molecules^32,45 or only less than 30% (Sillen A, Lippens G, unpublished data) suggests that other poly anions might be better suited to prepare fibers for structural biology. Careful dosing and characterization of the fibers through a combination of biochemical and low resolution spectroscopic methods will thereby be one of the corner stones for the structural elucidation of the PHF core region.Can phosphorylation lead to an equivalent charge compensation mechanisms, or are other structural factors in play? The AD P-Tau was shown to form filaments upon incubation at physiologically relevant conditions.⁴⁶ Upon dephosphorylation, however, all isoforms loose this capacity, suggesting that time wise, phosphorylation comes before aggregation. However, the same study revealed that the three or four microtubule binding domains cannot be phosphorylated for aggregation to occur, limiting the role of phosphorylation to charge compensation of the inhibitory regions up- and downstream of the MTBRs. Although in agreement with the finding that the isolated MTBRs aggregate more readily than the full length protein, this seems in contradiction with the aggregation model induced by exogeneous poly anions (Fig. 2), where charge compensation is not limited to the MTBR flanking regions, but also within the repeats itself. We are presently working with recombinant Tau and different kinases to reproduce the aggregation without additional poly anions. This should allow us to apply our structural biology tools to the fiber formation induced by the sole event of phosphorylation, and hence get better insight in the physiological aggregation mechanism. At the same time, we hope to get insight in the important but unanswered question to what certain AD specific antibodies really detect. Indeed, some of these have been classified as “conformational antibodies.” Structural elucidation of the antibody in complex with its phosphorylated Tau antigen could be a major step forward in the understanding of the distinguishing features of PHF-Tau, and hence in the aggregation mechanism.Open in a separate windowFigure 2(Left) Electron microscopy picture of a Tau PHF promoted by incubation with heparin. (Right) Model for the amyloid core of the fiber, with heparin providing for the negative charges essential to compensate for the positive stretch formed by parallel in register stacking of lysine containing peptides.³³Phosphorylation is evidently not only a signal for aggregation, but is present in very many physiological processes. Just to mention a few aspects concerning Tau, it promotes equally the interaction with Hsc70, which acts as a linker of the CHIP E3 ligase, thereby establishing a link with its degradation.⁴⁷ Other protein components such as the prolyl cis/trans isomerase Pin1 equally interact with the phosphorylated Tau, and might even restore its incapacity of microtubule formation.⁴⁸ Very recently, phosphorylated Tau was shown to interact with actin filaments, thereby influencing their bundling and association into so-called Hirano bodies.⁴⁹ A detailed biochemical/biophysical study of these different pathways might lead to a better understanding of Tau''s role in Alzheimer''s disease, and hopefully open a novel therapeutic window on this disease.     

Neuroprotective Agents: Is Effective on Toxicity in Glial Cells?

1. Glial cells are the most abundant cell population in the central nervous system. The aim of this study was to examine the effects of melatonin, 7-nitroindazole, and riluzole on glutamate toxicity in primary glial cell culture. 2. Glutamate toxicity was induced by addition of 100 μM glutamate to the cultures, and then 100 μM melatonin, 500 μM 7-nitroindazole, and 10 (M riluzole were administered in different groups. Lactate Dehydrogenase activity and nitrite levels were determined at the 1st, 6th, and 24th h. 3. Melatonin, 7-nitroindazole, and riluzole decrease Lactate Dehydrogenase activity at the 1st, 6th, and 24th h (at all hours, p<0.05). Nitrite levels were decreased by melatonin and riluzole at the 1st, 6th, and 24th h. 4. In this study, we observed that melatonin, 7-nitroindazole, and riluzole are effective as protective agents on glutamate toxicity in mixed glial cells.     

IFNα Serum Levels Are Associated with Endothelial Progenitor Cells Imbalance and Disease Features in Rheumatoid Arthritis Patients

Introduction

IFNα has been largely implicated in the ethiopathogenesis of autoimmune diseases but only recently it has been linked to endothelial damage and accelerated atherosclerosis in autoimmunity. In addition, proinflammatory conditions are supposed to be implicated in the cardiovascular status of these patients. Since a role for IFNα in endothelial damage and impaired Endothelial Progenitor Cell (EPC) number and function has been reported in other diseases, we aimed to evaluate the potential associations of IFNα serum levels on EPC populations and cytokine profiles in Rheumatoid Arthritis (RA) patients.

Methods

pre-EPC, EPC and mature EPC (mEPC) populations were quantified by flow cytometry analyzing their differential CD34, CD133 and VEGFR2 expression in blood samples from 120 RA patients, 52 healthy controls (HC), and 83 systemic lupus erythematosus (SLE) patients as disease control. Cytokine serum levels were measured by immunoassays and clinical and immunological data, including cardiovascular (CV) events and CV risk factors, were retrospectively obtained by reviewing clinical records.

Results

Long-standing, but not recent onset RA patients displayed a significant depletion of all endothelial progenitor populations, unless high IFNα levels were present. In fact, the IFN^high RA patient group (n = 40, 33%), showed increased EPC levels, comparable to SLE patients. In addition, high IFNα serum levels were associated with higher disease activity (DAS28), presence of autoantibodies, higher levels of IL-1β, IL-6, IL-10 and MIP-1α, lower amounts of TGF-β, and increased mEPC/EPC ratio, thus suggesting higher rates of endothelial damage and an endothelial repair failure. Finally, the relationship between high IFNα levels and occurrence of CV events observed in RA patients seems to support this hypothesis.

Conclusions

IFNα serum marker could be used to identify a group of RA patients with increased disease activity, EPC imbalance, enhanced proinflammatory profile and higher cardiovascular risk, probably due, at least in part, to an impaired endothelial repair.     

Molecular Probes: What Is the Range of Their Interaction with the Environment?

We performed pressure-tuning hole-burning experiments on a modified cytochrome c protein in a glycerol/buffer glass. The shift and the broadening of the holes were investigated for various frequencies within the inhomogeneous band. On the basis of a simple model, we were able to estimate the interaction range between chromophore and protein. It is ~4.5 Å. The parameters that enter the model are the compressibility, the static mean-square displacement, the inhomogeneous width, and the average spectral shift per pressure. From this result and from our experiments on pressure-induced denaturing, we conclude that water molecules have to be brought very close to the chromophore during the denaturation process.     

Hypoxia-Inducible Factor-2α Is an Essential Catabolic Regulator of Inflammatory Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disorder that manifests as chronic inflammation and joint tissue destruction. However, the etiology and pathogenesis of RA have not been fully elucidated. Here, we explored the role of the hypoxia-inducible factors (HIFs), HIF-1α (encoded by HIF1A) and HIF-2α (encoded by EPAS1). HIF-2α was markedly up-regulated in the intimal lining of RA synovium, whereas HIF-1α was detected in a few cells in the sublining and deep layer of RA synovium. Overexpression of HIF-2α in joint tissues caused an RA-like phenotype, whereas HIF-1α did not affect joint architecture. Moreover, a HIF-2α deficiency in mice blunted the development of experimental RA. HIF-2α was expressed mainly in fibroblast-like synoviocytes (FLS) of RA synovium and regulated their proliferation, expression of RANKL (receptor activator of nuclear factor–κB ligand) and various catabolic factors, and osteoclastogenic potential. Moreover, HIF-2α–dependent up-regulation of interleukin (IL)-6 in FLS stimulated differentiation of T_H17 cells—crucial effectors of RA pathogenesis. Additionally, in the absence of IL-6 (Il6 ^−/− mice), overexpression of HIF-2α in joint tissues did not cause an RA phenotype. Thus, our results collectively suggest that HIF-2α plays a pivotal role in the pathogenesis of RA by regulating FLS functions, independent of HIF-1α.     

Astrogliosis in CNS Pathologies: Is There A Role for Microglia?

Astrogliosis, a cellular reaction with specific structural and functional characteristics, represents a remarkably homotypic response of astrocytes to all kinds of central nervous system (CNS) pathologies. Astrocytes play diverse functions in the brain, both harmful and beneficial. Mounting evidence indicates that astrogliosis is an underlying component of a diverse range of diseases and associated neuropathologies. The mechanisms that lead to astrogliosis are not fully understood, nevertheless, damaged neurons have long been reported to induce astrogliosis and astrogliosis has been used as an index for underlying neuronal damage. As the predominant source of proinflammatory factors in the CNS, microglia are readily activated under certain pathological conditions. An increasing body of evidence suggests that release of cytokines and other soluble products by activated microglia can significantly influence the subsequent development of astrogliosis and scar formation in CNS. It is well known that damaged neurons activate microglia very quickly, therefore, it is possible that activated microglia contribute factors/mediators through which damaged neuron induce astrogliosis. The hypothesis that activated microglia initiate and maintain astrogliosis suggests that suppression of microglial overactivation might effectively attenuate reactive astrogliosis. Development of targeted anti-microglial activation therapies might slow or halt the progression of astrogliosis and, therefore, help achieve a more beneficial environment in various CNS pathologies.     

Neo-Epitopes—Fragments of Cartilage and Connective Tissue Degradation in Early Rheumatoid Arthritis and Unclassified Arthritis

Objective

Tissue destruction in rheumatoid arthritis (RA) is predominantly mediated by matrix metalloproteinases (MMPs), thereby generating protein fragments. Previous studies have revealed that these fragments include MMP-mediated collagen type I, II, and III degradation, citrullinated and MMP-degraded vimentin and MMP degraded C-reactive protein. We evaluated if biomarkers measuring serum levels of specific sequences of the mentioned fragments would provide further information of diagnostic and/or prognostic processes in early arthritis.

Methods

Ninety-two early arthritis patients (arthritis duration<1 year, DMARD naïve) were enrolled. Patients either fulfilled the ACR/EULAR2010 criteria for RA (n = 60) or had unclassified arthritis (UA) (n = 32). Patients fulfilling the RA criteria after 2 years follow-up were classified into non-erosive (n = 25), or erosive disease (n = 13). Concentrations of the biomarkers: C1M, C2M, C3M, VICM and CRPM were measured in baseline serum.

Results

C1M, C3M and CRPM were able to discriminate between the UA and RA baseline diagnosis in 92 patients with an AUROC of 0.64 (95%CI 0.517 to 0.762), 0.73 (95%CI 0.622 to 0.838) and 0.68 (95%CI 0.570 to 0.795). C2M showed a potential for discrimination between non-erosive and erosive disease in 38 patients with an AUROC of 0.75 (95%CI 0.597 to 0.910). All of the applied biomarkers correlated with one or more of the disease activity parameters: DAS28, ESR, CRP, SJC66, TJC68 and/or HAQ.

Conclusion

This is the first study evaluating the applied biomarkers at this early stage of arthritis. C1M, C3M, CRPM might be the best diagnostic marker, whereas high levels of C2M indicated progression of disease at follow-up in early RA patients.     

Is Helicobacter pylori a True Microaerophile?

BACKGROUND: There is no general consensus about the specific oxygen and carbon dioxide requirements of the human pathogen Helicobacter pylori. This bacterium is considered a microaerophile and consequently, it is grown under atmospheres at oxygen tensions 5-19% and carbon dioxide tensions 5-10%, both for clinical and basic and applied research purposes. The current study compared the growth of H. pylori in vitro, under various gas atmospheres, and determined some specific changes in the physiology of bacteria grown under different oxygen partial pressures. METHODS: Measurements of bacterial growth under various conditions were carried out employing classical solid and liquid culture techniques. Enzymatic activities were measured using spectrophotometric assays. RESULTS: H. pylori and all the other Helicobacter spp. tested had an absolute requirement for elevated carbon dioxide concentrations in the growth atmosphere. In contrast with other Helicobacter spp., H. pylori can tolerate elevated oxygen tensions when grown at high bacterial concentrations. Under 5% CO(2), the bacterium showed similar growth in liquid cultures under oxygen tensions from microaerobic (< 5%) to fully aerobic (21%) at cell densities higher than 5 x 10(5) cfu/ml for media supplemented with horse serum and 5 x 10(7) cfu/ml for media supplemented with beta-cyclodextrin. Evidence that changes occurred in the physiology of H. pylori was obtained by comparing the activities of ferredoxin:NADH (nicotinamide adenine dinucleotide) oxidoreductases of bacteria grown under microaerobic and aerobic atmospheres. CONCLUSIONS: H. pylori is a capnophile able to grow equally well in vitro under microaerobic or aerobic conditions at high bacterial concentrations, and behaved like oxygen-sensitive microaerophiles at low cell densities. Some characteristics of H. pylori cells grown in vitro under microaerobic conditions appeared to mimic better the physiology of organisms grown in their natural niche in the human stomach.     

Genomic Heritability: What Is It?

Whole-genome regression methods are being increasingly used for the analysis and prediction of complex traits and diseases. In human genetics, these methods are commonly used for inferences about genetic parameters, such as the amount of genetic variance among individuals or the proportion of phenotypic variance that can be explained by regression on molecular markers. This is so even though some of the assumptions commonly adopted for data analysis are at odds with important quantitative genetic concepts. In this article we develop theory that leads to a precise definition of parameters arising in high dimensional genomic regressions; we focus on the so-called genomic heritability: the proportion of variance of a trait that can be explained (in the population) by a linear regression on a set of markers. We propose a definition of this parameter that is framed within the classical quantitative genetics theory and show that the genomic heritability and the trait heritability parameters are equal only when all causal variants are typed. Further, we discuss how the genomic variance and genomic heritability, defined as quantitative genetic parameters, relate to parameters of statistical models commonly used for inferences, and indicate potential inferential problems that are assessed further using simulations. When a large proportion of the markers used in the analysis are in LE with QTL the likelihood function can be misspecified. This can induce a sizable finite-sample bias and, possibly, lack of consistency of likelihood (or Bayesian) estimates. This situation can be encountered if the individuals in the sample are distantly related and linkage disequilibrium spans over short regions. This bias does not negate the use of whole-genome regression models as predictive machines; however, our results indicate that caution is needed when using marker-based regressions for inferences about population parameters such as the genomic heritability.