Low sclerostin levels: a predictive marker of persistent inflammation in ankylosing spondylitis during anti-tumor necrosis factor therapy?

Introduction

Sclerostin levels have been reported to be low in ankylosing spondylitis (AS), but there is no data regarding the possible role of this Wnt inhibitor during anti-tumor necrosis factor (TNF) therapy. The present study longitudinally evaluated sclerostin levels, inflammatory markers and bone mineral density (BMD) in AS patients under anti-TNF therapy.

Methods

Thirty active AS patients were assessed at baseline, 6 and 12 months after anti-TNF therapy regarding clinical parameters, inflammatory markers, BMD and baseline radiographic damage (mSASSS). Thirty age- and sex-matched healthy individuals comprised the control group. Patients'' sclerostin levels, sclerostin binding low-density lipoprotein receptor-related protein 6 (LRP6) and BMD were evaluated at the same time points and compared to controls.

Results

At baseline, AS patients had lower sclerostin levels (60.5 ± 32.7 vs. 96.7 ± 52.9 pmol/L, P = 0.002) and comparable sclerostin binding to LRP6 (P = 0.387) than controls. Improvement of Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), Bath Ankylosing Spondylitis Metrology Index (BASMI), Ankylosing Spondylitis quality of life (ASQoL) was observed at baseline vs. 6 vs. 12 months (P < 0.01). Concomitantly, a gradual increase in spine BMD (P < 0.001) and a positive correlation between baseline mSASSS and spine BMD was found (r = 0.468, P < 0.01). Inflammatory parameters reduction was observed comparing baseline vs. 6 vs. 12 months (P <0.01). Sclerostin levels progressively increased [baseline (60.5 ± 32.7) vs. 6 months (67.1 ± 31.9) vs. 12 months (72.7 ± 32.3) pmol/L, P <0.001]. At 12 months, the sclerostin levels remained significantly lower in patients compared to controls (72.7 ± 32.3 vs. 96.70 ± 52.85 pmol/L, P = 0.038). Moreover, sclerostin serum levels at 12 months were lower in the 10 patients with high C reactive protein (CRP) (≥ 5 mg/l) compared to the other 20 patients with normal CRP (P = 0.004). Of note, these 10 patients with persistent inflammation also had lower sclerostin serum levels at baseline compared to the other patients (P = 0.023). Univariate logistic regression analysis demonstrated that AS patients with lower sclerostin serum levels had an increased risk to have high CRP at 12 months (odds ratio = 7.43, 95% CI 1.23 to 45.01, P = 0.020) than those with higher sclerostin values.

Conclusions

Persistent low sclerostin levels may underlie continuous inflammation in AS patients under anti-TNF therapy.     

Frequency of infection of Lutzomyia phlebotomines with Leishmania braziliensis in a Brazilian endemic area as assessed by pinpoint capture and polymerase chain reaction

Leishmania infected of Lutzomyia spp. are rare in endemic areas. We tested the hypothesis that there is clustering of infected vectors by combining pinpoint capture with sensitive L. braziliensis kDNA minicircle specific PCR/dot blot in an endemic area in the State of Bahia. Thirty out of 335 samples (10 to 20 sand flies/sample; total of 4,027 female sand flies) were positive by PCR analysis and dot blot leading to a underestimated overall rate of 0.4% positive phlebotomines. However, 83.3% of the positive samples were contributed by a single sector out of four sectors of the whole studied area. This resulted in a rate of 1.5% Leishmania positive phlebotomines for this sector, far above rates of other sectors. Incidence of American cutaneous leishmaniasis cases for this sector was about twice that for other sectors. Our results show that there is a non-homogeneous distribution of Leishmania-infected vectors. Such a clustering may have implications in control strategies against leishmaniasis, and reinforces the necessity of understanding the ecological and geographical factors involved in leishmanial transmission.     

Design and implementation of microarray gene expression markup language (MAGE-ML)

Background

Meaningful exchange of microarray data is currently difficult because it is rare that published data provide sufficient information depth or are even in the same format from one publication to another. Only when data can be easily exchanged will the entire biological community be able to derive the full benefit from such microarray studies.

Results

To this end we have developed three key ingredients towards standardizing the storage and exchange of microarray data. First, we have created a minimal information for the annotation of a microarray experiment (MIAME)-compliant conceptualization of microarray experiments modeled using the unified modeling language (UML) named MAGE-OM (microarray gene expression object model). Second, we have translated MAGE-OM into an XML-based data format, MAGE-ML, to facilitate the exchange of data. Third, some of us are now using MAGE (or its progenitors) in data production settings. Finally, we have developed a freely available software tool kit (MAGE-STK) that eases the integration of MAGE-ML into end users' systems.

Conclusions

MAGE will help microarray data producers and users to exchange information by providing a common platform for data exchange, and MAGE-STK will make the adoption of MAGE easier.     

Integrin alpha IIb beta 3-dependent calcium signals regulate platelet-fibrinogen interactions under flow. Involvement of phospholipase C gamma 2

Platelet adhesion to fibrinogen is important for platelet aggregation and thrombus growth. In this study we have examined the mechanisms regulating platelet adhesion on immobilized fibrinogen under static and shear conditions. We demonstrate that integrin alpha IIb beta 3 engagement of immobilized fibrinogen is sufficient to induce an oscillatory calcium response, necessary for lamellipodial formation and platelet spreading. Released ADP increases the proportion of platelets exhibiting a cytosolic calcium response but is not essential for calcium signaling or lamellipodial extension. Pretreating platelets with the Src kinase inhibitor PP2, the inositol 1,4,5-trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate (APB-2), or the phospholipase C (PLC) inhibitor U73122 abolished calcium signaling and platelet spreading, suggesting a major role for Src kinase-regulated PLC isoforms in these processes. Analysis of PLC gamma 2-/- mouse platelets revealed a major role for this isoform in regulating cytosolic calcium flux and platelet spreading on fibrinogen. Under flow conditions, platelets derived from PLC gamma 2-/- mice formed less stable adhesive interactions with fibrinogen, particularly in the presence of ADP antagonists. Our studies define an important role for PLC gamma 2 in integrin alpha IIb beta 3-dependent calcium flux, necessary for stable platelet adhesion and spreading on fibrinogen. Furthermore, they establish an important cooperative signaling role for PLC gamma 2 and ADP in regulating platelet adhesion efficiency on fibrinogen.     

pH dependence of the dissociation of multimeric hemoglobin probed by high hydrostatic pressure

We investigated the thermodynamic features of the classic alkaline dissociation of multimeric hemoglobin (3.1 MDa) from Glossoscolex paulistus (Annelidea) using high hydrostatic pressure. Light scattering measurements up to microscopic thermodynamic equilibrium indicated a high pH dependency of dissociation and association. Electron microscopy and gel filtration corroborated these findings. The volume change of dissociation decreased in absolute values from -48.0 mL/mol of subunit at pH 6.0 to -19.2 mL/mol at pH 9.0, suggesting a lack of protein interactions under alkaline conditions. Concomitantly, an increase in pH reduced the Gibbs free energy of dissociation from 37.7 to 27.5 kJ/mol of subunit. The stoichiometry of proton release calculated from the pressure-induced dissociation curves was +0.602 mol of H(+)/mol of subunit. These results provide a direct quantification of proton participation in stabilizing the aggregated state of the hemoglobin, and contribute to our understanding of protein-protein interactions and of the surrounding conditions that modulate the process of aggregation.     

Comparative Metabolomic Profiling Reveals That Dysregulated Glycolysis Stemming from Lack of Salvage NAD+ Biosynthesis Impairs Reproductive Development in Caenorhabditis elegans

Temporal developmental progression is highly coordinated in Caenorhabditis elegans. However, loss of nicotinamidase PNC-1 activity slows reproductive development, uncoupling it from its typical progression relative to the soma. Using LC/MS we demonstrate that pnc-1 mutants do not salvage the nicotinamide released by NAD⁺ consumers to resynthesize NAD⁺, resulting in a reduction in global NAD⁺ bioavailability. We manipulate NAD⁺ levels to demonstrate that a minor deficit in NAD⁺ availability is incompatible with a normal pace of gonad development. The NAD⁺ deficit compromises NAD⁺ consumer activity, but we surprisingly found no functional link between consumer activity and reproductive development. As a result we turned to a comparative metabolomics approach to identify the cause of the developmental phenotype. We reveal widespread metabolic perturbations, and using complementary pharmacological and genetic approaches, we demonstrate that a glycolytic block accounts for the slow pace of reproductive development. Interestingly, mitochondria are protected from both the deficiency in NAD⁺ biosynthesis and the effects of reduced glycolytic output. We suggest that compensatory metabolic processes that maintain mitochondrial activity in the absence of efficient glycolysis are incompatible with the requirements for reproductive development, which requires high levels of cell division. In addition to demonstrating metabolic requirements for reproductive development, this work also has implications for understanding the mechanisms behind therapeutic interventions that target NAD⁺ salvage biosynthesis for the purposes of inhibiting tumor growth.     

(R)-NODAGA-PSMA: A Versatile Precursor for Radiometal Labeling and Nuclear Imaging of PSMA-Positive Tumors

Purpose

The present study aims at developing and evaluating an urea-based prostate specific membrane antigen (PSMA) inhibitor suitable for labeling with ¹¹¹In for SPECT and intraoperative applications as well as ⁶⁸Ga and ⁶⁴Cu for PET imaging.

Methods

The PSMA-based inhibitor-lysine-urea-glutamate-coupled to the spacer Phe-Phe-D-Lys(suberoyl) and functionalized with the enantiomerically pure prochelator (R)-1-(1-carboxy-3-carbotertbutoxypropyl)-4,7-carbotartbutoxymethyl)-1,4,7-triazacyclononane ((R)-NODAGA(tBu)₃), to obtain (R)-NODAGA-Phe-Phe-D-Lys(suberoyl)-Lys-urea-Glu (CC34). CC34 was labeled with ¹¹¹In, ⁶⁸Ga and ⁶⁴Cu. The radioconjugates were further evaluated in vitro and in vivo in LNCaP xenografts by biodistribution and PET studies. Biodistribution studies were also performed with ⁶⁸Ga-HBED-CC-PSMA (HBED-CC: N,N′-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N′-diacetic acid) and ¹¹¹In-PSMA-617 for comparison.

Results

⁶⁸Ga-CC34, ⁶⁴Cu-CC34, and ¹¹¹In-CC34 were prepared in radiochemical purity >95%. ^68/natGa-CC34, ^64/natCu-CC34, ^111/natIn-CC34, ^68/natGa-HBED-CC-PSMA, and ^111/natIn-PSMA-617 exhibited high affinity for the LNCaP cells, with K_d values of 19.3±2.5 nM, 27.5±2.7 nM, 5.5±0.9 nM, 2.9±0.6 nM and 5.4±0.8 nM, respectively. They revealed comparable internalization profiles with approximately 75% of the total cell associated activity internalized after 3 h of incubation. ⁶⁸Ga-CC34 showed very high stability after its administration in mice. Tumor uptake of ⁶⁸Ga-CC34 (14.5±2.9% IA/g) in LNCaP xenografts at 1 h p.i. was comparable to ⁶⁸Ga-HBED-CC-PSMA (15.8±1.4% IA/g) (P = 0.67). The tumor-to-normal tissue ratios at 1 and 2 h p.i of ⁶⁸Ga-CC34 were also comparable to ⁶⁸Ga-HBED-CC-PSMA (P>0.05). Tumor uptake of ¹¹¹In-CC34 (28.5±2.6% IA/g) at 1 h p.i. was lower than ¹¹¹In-PSMA-617 (52.1±6.5% IA/g) (P = 0.02). The acquisition of PET-images with ⁶⁴Cu-CC34 at later time points showed wash-out from the kidneys, while tumor uptake still remained relatively high. This resulted in an increased tumor-to-kidney ratio over time.

Conclusions

⁶⁸Ga-CC34 is comparable to ⁶⁸Ga-HBED-CC-PSMA in terms of tumor uptake and tumor to normal tissue ratios. ⁶⁴Cu-CC34 could enable high contrast imaging of PSMA positive tissues characterized by elevated expression of PSMA or when delayed imaging is required. ⁶⁴Cu-CC34 is currently being prepared for clinical translation.