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1.  What is the future of targeted therapy in rheumatology: biologics or small molecules? 
BMC Medicine  2014;12:43.
Background
Until late in the 20th century, the therapy of rheumatic diseases relied on the use of drugs that had been developed through empirical approaches without detailed understanding of the molecular mechanisms involved. That approach changed with the introduction of biologic therapeutics at the end of the 20th century and by the recent development of small-molecule inhibitors of intracellular signal transduction pathways. Here we compare and discuss the advantages and disadvantages of those two groups of targeted anti-inflammatory therapeutics.
Discussion
TNF-blocking biologic agents were introduced into the therapy of rheumatoid arthritis and other autoimmune and inflammatory diseases in the late 1990s. Further biologic agents targeting cytokine networks or specific lymphocyte subsets have since been added to the armamentarium of anti-rheumatic therapy. During the last few years, another wave of novel discoveries led to the development of a new class of small molecule anti-inflammatory compounds targeting intracellular signal transduction molecules, such as tyrosine kinases. In all those cases, the specific targets of the drugs are well defined and significant knowledge about their role in the disease pathomechanism is available, qualifying them for being targeted therapeutics for inflammatory rheumatic diseases. While both groups of targeted therapeutics offer significant clinical benefit, they clearly differ in several aspects, such as the localization of their targets, their route of administration and target specificity, as well as technical details such as manufacturing procedures and cost basis. In this debate paper, we compare the advantages and disadvantages of the two different approaches, aiming to shed light on the possible future of targeted therapies.
Summary
Biologic therapeutics and small-molecule inhibitors both have significant advantages and disadvantages in the therapy of rheumatic diseases. The future of targeted therapies is one of the most exciting questions of current rheumatology research and therapy.
doi:10.1186/1741-7015-12-43
PMCID: PMC3975154  PMID: 24620738
Rheumatoid arthritis; Biologic therapies; TNF antagonists; Small molecule therapeutics; Kinase inhibitors; Tofacitinib
2.  Higher Levels of Autoantibodies Targeting Mutated Citrullinated Vimentin in Patients with Psoriatic Arthritis Than in Patients with Psoriasis Vulgaris 
Antibodies against citrullinated proteins/peptides (ACPAs), and especially antibodies targeting mutated citrullinated vimentin (anti-MCVs), are novel biomarkers of rheumatoid arthritis (RA). Whereas ACPAs are specific and sensitive markers for RA, there have hardly been any reports relating to ACPAs in psoriatic arthritis (PsA) or in psoriasis without joint symptoms (PsO). The aim of the present study was to investigate the prevalence of anti-MCVs in PsA and PsO. Serum anti-MCV titers were measured in 46 PsA and 42 PsO patients and in 40 healthy controls by means of a commercial enzyme-linked immunosorbent assay. The potential correlations of the serum autoantibody levels with several clinical and laboratory parameters were examined. The anti-MCV levels in the PsA patients were significantly higher than those in the PsO group. Among the clinical variables, the presence of tender knee joints and nail psoriasis was significantly associated with anti-MCV positivity in the PsA patients. Higher anti-MCV titers in the PsO patients were associated with a more severe disease course and with the early onset of psoriatic skin symptoms. Our results suggest that anti-MCVs can be used as novel markers in the diagnosis of PsA and in a subset of PsO patients.
doi:10.1155/2013/474028
PMCID: PMC3614022  PMID: 23573111
3.  Fine Mapping and Conditional Analysis Identify a New Mutation in the Autoimmunity Susceptibility Gene BLK that Leads to Reduced Half-Life of the BLK Protein 
Annals of the Rheumatic Diseases  2012;71(7):1219-1226.
Objectives
To perform fine mapping of the autoimmunity susceptibility gene BLK and identify functional variants involved in SLE.
Methods
Genotyping of 1163 European SLE patients and 1482 controls and imputation were performed covering the BLK gene with 158 SNPs. Logistic regression analysis was done using PLINK and conditional analyses using GENABEL’s test score. Transfections of BLK constructs on HEK293 cells containing the novel mutation or the wild-type form were made to analyse their effect on protein half-life using a protein stability assay, cycloheximide and Western blot. CHiP-qPCR for NFkB binding.
Results
Fine mapping of BLK identified two independent genetic effects with functional consequences: one represented by two tightly linked associated haplotype blocks significantly enriched for NFκB-binding sites and numerous putative regulatory variants whose risk alleles correlated with low BLK mRNA levels. Binding of NFkBp50 and p65 to an associated 1.2Kb haplotype segment was confirmed. A second independent genetic effect was represented by an Ala71Thr, low-frequency missense substitution with an OR = 2.31 (95% c.i. 1.38–3.86). The 71Thr decreased BLK protein half-life.
Conclusions
Our results show that rare and common regulatory variants in BLK are involved in disease susceptibility and both, albeit independently lead to reduced levels of BLK protein.
doi:10.1136/annrheumdis-2011-200987
PMCID: PMC3375585  PMID: 22696686
systemic lupus erythematosus; autoimmunity; genetics; polymorphism; B-cells; autoantibodies; B-lymphocyte tyrosine kinase
4.  Association between autoimmune pancreatitis and systemic autoimmune diseases 
AIM: To investigate the association between autoimmune pancreatitis (AIP) and systemic autoimmune diseases (SAIDs) by measurement of serum immunoglobulin G4 (IgG4).
METHODS: The serum level of IgG4 was measured in 61 patients with SAIDs of different types who had not yet participated in glucocorticosteroid treatment. Patients with an elevated IgG4 level were examined by abdominal ultrasonography (US) and, in some cases, by computer tomography (CT).
RESULTS: Elevated serum IgG4 levels (919 ± 996 mg/L) were detected in 17 (28%) of the 61 SAID patients. 10 patients had Sjögren’s syndrome (SS) (IgG4: 590 ± 232 mg/L), 2 of them in association with Hashimoto’s thyroiditis, and 7 patients (IgG4: 1388 ± 985.5 mg/L) had systemic lupus erythematosus (SLE). The IgG4 level in the SLE patients and that in patients with SS were not significantly different from that in AIP patients (783 ± 522 mg/L). Abdominal US and CT did not reveal any characteristic features of AIP among the SAID patients with an elevated IgG4 level.
CONCLUSION: The serum IgG4 level may be elevated in SAIDs without the presence of AIP. The determination of serum IgG4 does not seem to be suitable for the differentiation between IgG4-related diseases and SAIDs.
doi:10.3748/wjg.v18.i21.2649
PMCID: PMC3370001  PMID: 22690073
Autoimmune pancreatitis; Serum immunoglobulin G4 level; Systemic lupus erythematosus; Sjögren’s syndrome; Mikulicz’s disease
5.  Adaptive Immunity in Ankylosing Spondylitis: Phenotype and Functional Alterations of T-Cells before and during Infliximab Therapy 
Our aim was to assess the phenotype of T-cell subsets in patients with ankylosing spondylitis (AS), a chronic inflammatory rheumatic disease. In addition, we also tested short-term T-cell activation characteristics. Measurements were done in 13 AS patients before and during the intravenous therapy with anti-TNF agent infliximab (IFX). Flow cytometry was used to determine T-cell subsets in peripheral blood and their intracellular signaling during activation. The prevalence of Th2 and Th17 cells responsible for the regulation of adaptive immunity was higher in AS than in 9 healthy controls. Although IFX therapy improved patients' condition, immune phenotype did not normalize. Cytoplasmic and mitochondrial calcium responses of CD4+ and CD8+ cells to a specific activation were delayed, while NO generation was increased in AS. NO generation normalized sooner upon IFX than calcium response. These results suggest an abnormal immune phenotype with functional disturbances of CD4+ and CD8+ cells in AS.
doi:10.1155/2012/808724
PMCID: PMC3182565  PMID: 21969839
6.  Molecular Evolution of Phosphoprotein Phosphatases in Drosophila 
PLoS ONE  2011;6(7):e22218.
Phosphoprotein phosphatases (PPP), these ancient and important regulatory enzymes are present in all eukaryotic organisms. Based on the genome sequences of 12 Drosophila species we traced the evolution of the PPP catalytic subunits and noted a substantial expansion of the gene family. We concluded that the 18–22 PPP genes of Drosophilidae were generated from a core set of 8 indispensable phosphatases that are present in most of the insects. Retropositons followed by tandem gene duplications extended the phosphatase repertoire, and sporadic gene losses contributed to the species specific variations in the PPP complement. During the course of these studies we identified 5, up till now uncharacterized phosphatase retrogenes: PpY+, PpD5+, PpD6+, Pp4+, and Pp6+ which are found only in some ancient Drosophila. We demonstrated that all of these new PPP genes exhibit a distinct male specific expression. In addition to the changes in gene numbers, the intron-exon structure and the chromosomal localization of several PPP genes was also altered during evolution. The G−C content of the coding regions decreased when a gene moved into the heterochromatic region of chromosome Y. Thus the PPP enzymes exemplify the various types of dynamic rearrangements that accompany the molecular evolution of a gene family in Drosophilidae.
doi:10.1371/journal.pone.0022218
PMCID: PMC3137614  PMID: 21789237
7.  Digalactosyl-diacylglycerol-deficiency lowers the thermal stability of thylakoid membranes 
Photosynthesis Research  2010;105(3):229-242.
We investigated the effects of digalactosyl-diacylglycerol (DGDG) on the organization and thermal stability of thylakoid membranes, using wild-type Arabidopsis thaliana and the DGDG-deficient mutant, dgd1. Circular-dichroism measurements reveal that DGDG-deficiency hampers the formation of the chirally organized macrodomains containing the main chlorophyll a/b light-harvesting complexes. The mutation also brings about changes in the overall chlorophyll fluorescence lifetimes, measured in whole leaves as well as in isolated thylakoids. As shown by time-resolved measurements, using the lipophylic fluorescence probe Merocyanine 540 (MC540), the altered lipid composition affects the packing of lipids in the thylakoid membranes but, as revealed by flash-induced electrochromic absorbance changes, the membranes retain their ability for energization. Thermal stability measurements revealed more significant differences. The disassembly of the chiral macrodomains around 55°C, the thermal destabilization of photosystem I complex at 61°C as detected by green gel electrophoresis, as well as the sharp drop in the overall chlorophyll fluorescence lifetime above 45°C (values for the wild type—WT) occur at 4–7°C lower temperatures in dgd1. Similar differences are revealed in the temperature dependence of the lipid packing and the membrane permeability: at elevated temperatures MC540 appears to be extruded from the dgd1 membrane bilayer around 35°C, whereas in WT, it remains lipid-bound up to 45°C and dgd1 and WT membranes become leaky around 35 and 45°C, respectively. It is concluded that DGDG plays important roles in the overall organization of thylakoid membranes especially at elevated temperatures.
doi:10.1007/s11120-010-9581-5
PMCID: PMC2975056  PMID: 20645128
Arabidopsis mutants; Digalactosyl-diacylglycerol; dgd1 mutant; Thermal stability; Thylakoid lipids; Thylakoid membranes
8.  The powdery mildew resistance gene REN1 co-segregates with an NBS-LRR gene cluster in two Central Asian grapevines 
BMC Genetics  2009;10:89.
Background
Grape powdery mildew is caused by the North American native pathogen Erysiphe necator. Eurasian Vitis vinifera varieties were all believed to be susceptible. REN1 is the first resistance gene naturally found in cultivated plants of Vitis vinifera.
Results
REN1 is present in 'Kishmish vatkana' and 'Dzhandzhal kara', two grapevines documented in Central Asia since the 1920's. These cultivars have a second-degree relationship (half sibs, grandparent-grandchild, or avuncular), and share by descent the chromosome on which the resistance allele REN1 is located. The REN1 interval was restricted to 1.4 cM using 38 SSR markers distributed across the locus and the segregation of the resistance phenotype in two progenies of collectively 461 offspring, derived from either resistant parent. The boundary markers delimit a 1.4-Mbp sequence in the PN40024 reference genome, which contains 27 genes with known functions, 2 full-length coiled-coil NBS-LRR genes, and 9 NBS-LRR pseudogenes. In the REN1 locus of PN40024, NBS genes have proliferated through a mixture of segmental duplications, tandem gene duplications, and intragenic recombination between paralogues, indicating that the REN1 locus has been inherently prone to producing genetic variation. Three SSR markers co-segregate with REN1, the outer ones confining the 908-kb array of NBS-LRR genes. Kinship and clustering analyses based on genetic distances with susceptible cultivars representative of Central Asian Vitis vinifera indicated that 'Kishmish vatkana' and 'Dzhandzhal kara' fit well into local germplasm. 'Kishmish vatkana' also has a parent-offspring relationship with the seedless table grape 'Sultanina'. In addition, the distant genetic relatedness to rootstocks, some of which are derived from North American species resistant to powdery mildew and have been used worldwide to guard against phylloxera since the late 1800's, argues against REN1 being infused into Vitis vinifera from a recent interspecific hybridisation.
Conclusion
The REN1 gene resides in an NBS-LRR gene cluster tightly delimited by two flanking SSR markers, which can assist in the selection of this DNA block in breeding between Vitis vinifera cultivars. The REN1 locus has multiple layers of structural complexity compared with its two closely related paralogous NBS clusters, which are located some 5 Mbp upstream and 4 Mbp downstream of the REN1 interval on the same chromosome.
doi:10.1186/1471-2156-10-89
PMCID: PMC2814809  PMID: 20042081
9.  Effects of Dantrolene on Steps of Excitation-Contraction Coupling in Mammalian Skeletal Muscle Fibers 
The Journal of General Physiology  2001;118(4):355-376.
The effects of the muscle relaxant dantrolene on steps of excitation-contraction coupling were studied on fast twitch muscles of rodents. To identify the site of action of the drug, single fibers for voltage-clamp measurements, heavy SR vesicles for calcium efflux studies and solubilized SR calcium release channels/RYRs for lipid bilayer studies were isolated. Using the double Vaseline-gap or the silicone-clamp technique, dantrolene was found to suppress the depolarization-induced elevation in intracellular calcium concentration ([Ca2+]i) by inhibiting the release of calcium from the SR. The suppression of [Ca2+]i was dose-dependent, with no effect at or below 1 μM and a 53 ± 8% (mean ± SEM, n = 9, cut fibers) attenuation at 0 mV with 25 μM of extracellularly applied dantrolene. The drug was not found to be more effective if injected than if applied extracellularly. Calculating the SR calcium release revealed an equal suppression of the steady (53 ± 8%) and of the early peak component (46 ± 6%). The drug did not interfere with the activation of the voltage sensor in as much as the voltage dependence of both intramembrane charge movements and the L-type calcium currents (ICa) were left, essentially, unaltered. However, the inactivation of ICa was slowed fourfold, and the conductance was reduced from 200 ± 16 to 143 ± 8 SF−1 (n = 10). Dantrolene was found to inhibit thymol-stimulated calcium efflux from heavy SR vesicles by 44 ± 10% (n = 3) at 12 μM. On the other hand, dantrolene failed to affect the isolated RYR incorporated into lipid bilayers. The channel displayed a constant open probability for as long as 30–50 min after the application of the drug. These data locate the binding site for dantrolene to be on the SR membrane, but be distinct from the purified RYR itself.
PMCID: PMC2233700  PMID: 11585849
calcium current; intramembrane charge; calcium release; ryanodine receptor; single channel
10.  Fine mapping and conditional analysis identify a new mutation in the autoimmunity susceptibility gene BLK that leads to reduced half-life of the BLK protein 
Annals of the Rheumatic Diseases  2012;71(7):1219-1226.
Objectives
To perform fine mapping of the autoimmunity susceptibility gene BLK and identify functional variants involved in systemic lupus erythematosus (SLE).
Methods
Genotyping of 1163 European SLE patients and 1482 controls and imputation were performed covering the BLK gene with 158 single-nucleotide polymorphisms. Logistic regression analysis was done using PLINK and conditional analyses using GENABEL's test score. Transfections of BLK constructs on HEK293 cells containing the novel mutation or the wild type form were analysed for their effect on protein half-life using a protein stability assay, cycloheximide and western blot. CHiP-qPCR for detection of nuclear factor κ B (NFkB) binding.
Results
Fine mapping of BLK identified two independent genetic effects with functional consequences: one represented by two tightly linked associated haplotype blocks significantly enriched for NFκB-binding sites and numerous putative regulatory variants whose risk alleles correlated with low BLK mRNA levels. Binding of NFkBp50 and p65 to an associated 1.2 Kb haplotype segment was confirmed. A second independent genetic effect was represented by an Ala71Thr, low-frequency missense substitution with an OR=2.31 (95% CI 1.38 to 3.86). The 71Thr decreased BLK protein half-life.
Conclusions
These results show that rare and common regulatory variants in BLK are involved in disease susceptibility and both, albeit independently, lead to reduced levels of BLK protein.
doi:10.1136/annrheumdis-2011-200987
PMCID: PMC3375585  PMID: 22696686

Results 1-10 (10)