Low-dose oral imatinib in the treatment of systemic sclerosis interstitial lung disease unresponsive to cyclophosphamide: a phase II pilot study

Introduction

Pulmonary involvement represents a major cause of death of systemic sclerosis (SSc) patients. Recent data suggest that tyrosine kinase inhibitors, such as imatinib, may be a therapeutic option for SSc patients. However, preliminary published clinical trials were inconclusive about imatinib efficacy and showed side effects. The purpose of this study was to verify efficacy and tolerability of low-dose imatinib on interstitial lung disease in a cohort of SSc patients unresponsive to cyclophosphamide therapy.

Methods

Thirty consecutive SSc patients with active pulmonary involvement, unresponsive to cyclophosphamide, were treated with imatinib 200 mg/day for 6 months followed by a 6-month follow-up. A “good response” was defined as an increase of forced vital capacity (FVC) by more of 15% and/or increase of diffusing capacity of carbon monoxide (DL_CO) >15% and PaO₂ > 90% of initial value and high-resolution computed tomography (HRCT)-scan pattern unchanged or improved.

Results

Twenty-six patients completed the study. Three patients died and one patient was lost to follow-up. Four patients (15.32%) had a good response, 7 worsened and 15 had a stabilized lung disease. Overall, 19 (73.07%) patients had an improved or stabilized lung disease. After a 6-month follow-up, 12 (54.5%) of the 22 patients showed an improved or stabilized lung disease.

Conclusions

Lung function was stabilized in a large proportion of patients unresponsive to cyclophosphamide therapy and a beneficial outcome emerged from the analysis of HRCT lung scans. There was no significant improvement of skin involvement, and the low dose was well tolerated. These data provide useful suggestions to design future randomized clinical trials for SSc therapeutics.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00573326","term_id":"NCT00573326"}}NCT00573326. Registered 13 December 2007.     

The Lateral Membrane Organization and Dynamics of Myelin Proteins PLP and MBP Are Dictated by Distinct Galactolipids and the Extracellular Matrix

In the central nervous system, lipid-protein interactions are pivotal for myelin maintenance, as these interactions regulate protein transport to the myelin membrane as well as the molecular organization within the sheath. To improve our understanding of the fundamental properties of myelin, we focused here on the lateral membrane organization and dynamics of peripheral membrane protein 18.5-kDa myelin basic protein (MBP) and transmembrane protein proteolipid protein (PLP) as a function of the typical myelin lipids galactosylceramide (GalC), and sulfatide, and exogenous factors such as the extracellular matrix proteins laminin-2 and fibronectin, employing an oligodendrocyte cell line, selectively expressing the desired galactolipids. The dynamics of MBP were monitored by z-scan point fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS), while PLP dynamics in living cells were investigated by circular scanning FCS. The data revealed that on an inert substrate the diffusion rate of 18.5-kDa MBP increased in GalC-expressing cells, while the diffusion coefficient of PLP was decreased in sulfatide-containing cells. Similarly, when cells were grown on myelination-promoting laminin-2, the lateral diffusion coefficient of PLP was decreased in sulfatide-containing cells. In contrast, PLP''s diffusion rate increased substantially when these cells were grown on myelination-inhibiting fibronectin. Additional biochemical analyses revealed that the observed differences in lateral diffusion coefficients of both proteins can be explained by differences in their biophysical, i.e., galactolipid environment, specifically with regard to their association with lipid rafts. Given the persistence of pathological fibronectin aggregates in multiple sclerosis lesions, this fundamental insight into the nature and dynamics of lipid-protein interactions will be instrumental in developing myelin regenerative strategies.     

Effect of inositol hexakisphosphate on the EPR properties of the nitric oxide derivative of ferrous dromedary (Camelus dromedarius) hemoglobin. Evidence for two polyanion binding sites

The effect of inositol hexakisphosphate on the EPR properties of the nitric oxide derivative of ferrous dromedary (Camelus dromedarius) hemoglobin has been investigated at 110 K. In the absence of inositol hexakisphosphate, the nitrosyl derivative of dromedary hemoglobin shows an EPR spectrum with a rhombic shape and a weak hyperfine splitting in the gz region, a feature that is generally taken as characteristic of the high-affinity state of tetrameric hemoproteins. On addition of 1 mole of inositol hexakisphosphate/tetramer, three new hyperfine lines (Az = 1.7 mT), centered at gz = 2.01, appear; this type of spectrum is indicative of the low-affinity state of hemoglobins. A further addition of inositol hexakisphosphate, corresponding to a 20-fold molar excess, completely reverses the polyphosphate-dependent transition, giving an EPR spectrum that is exactly superimposable to that observed in the absence of the allosteric effector, i.e., is typical of the high-affinity state of the macromolecule. Both in the absence and presence of inositol hexakisphosphate, the EPR spectra are virtually independent of pH in the range explored (from 5.5 to 7.5). These results, taken together with the behavior of the nitric oxide derivative of human hemoglobin, provide further evidence for the existance in dromedary hemoglobin of two polyanion binding sites that affect in an opposite way the conformational equilibrium of the macromolecule.     

Impaired carotenogenesis can affect organization and functionality of etioplast membranes

The effects of impaired carotenogenesis on plastid membrane organization, functionality and stability were studied in etiolated barley plants grown at 20 and 30°C. The plants were treated with norflurazon or amitrole, two herbicides affecting phytoene desaturation and lycopene cyclization, respectively. At 20°C, the amitrole-treated etioplasts, which accumulated lycopene in their inner membranes, exhibited disorganized prolamellar bodies, containing a prevalent form of non-phototransformable protochlorophyllide (Pchlide). They also showed a certain difficulty in reducing the phototransformable pigment to chlorophyllide when exposed to light, and were unable to reform the active ternary complex [protochlorophyllide–oxidoreductase (POR)–Pchlide–NADPH] when placed back in darkness. No ultrastructural alterations were found in norflurazon-treated etioplasts, with carotenogenesis inhibited at the phytoene desaturation step. In these latter organelles, Pchlide, whose forms were comparable with those of the control etioplasts, was photoreduced quickly after illumination and the ternary complex was reformed during a subsequent dark period. Thus, the impaired carotenogenesis leading to the accumulation of lycopene showed greater interference with the etioplast membrane arrangement and functionality than did the earlier interruption of the biosynthetic pathway at the phytoene level. This might be due to the different interactions of the distinct carotenoid precursors with other membrane components. However, in etioplasts of norflurazon-treated plants, a rise in growth temperature caused a partial demolition of prolamellar bodies, showing a lowered thermostability of the carotenoid-deficient membranes. This latter effect strengthens the concept that a correct and complete carotenogenesis pathway, leading to the synthesis of polar carotenoids (i.e. xanthophylls), is required for the maintenance of stable plastid membranes.     

High‐Throughput DNA sequencing of ancient wood

Reconstructing the colonization and demographic dynamics that gave rise to extant forests is essential to forecasts of forest responses to environmental changes. Classical approaches to map how population of trees changed through space and time largely rely on pollen distribution patterns, with only a limited number of studies exploiting DNA molecules preserved in wooden tree archaeological and subfossil remains. Here, we advance such analyses by applying high‐throughput (HTS) DNA sequencing to wood archaeological and subfossil material for the first time, using a comprehensive sample of 167 European white oak waterlogged remains spanning a large temporal (from 550 to 9,800 years) and geographical range across Europe. The successful characterization of the endogenous DNA and exogenous microbial DNA of 140 (~83%) samples helped the identification of environmental conditions favouring long‐term DNA preservation in wood remains, and started to unveil the first trends in the DNA decay process in wood material. Additionally, the maternally inherited chloroplast haplotypes of 21 samples from three periods of forest human‐induced use (Neolithic, Bronze Age and Middle Ages) were found to be consistent with those of modern populations growing in the same geographic areas. Our work paves the way for further studies aiming at using ancient DNA preserved in wood to reconstruct the micro‐evolutionary response of trees to climate change and human forest management.     

Experimental and simulative dissociation of dimeric Cu,Zn superoxide dismutase doubly mutated at the intersubunit surface

The equilibrium properties of dimeric Photobacterium leiognathi Cu,Zn superoxide dismutase mutant bearing two negative charges in the amino acid clusters at the association interface has been studied, experimentally and computationally, and compared to those of the native enzyme. Pressure-dependent dissociation is observed for the mutant, as observed by the fluorescence shift of the unique tryptophan residue located at the intersubunit surface. The spectral shift occurs slowly, reaching a plateau after 15-20 min, and is fully reversible. Measurement of the degree of dissociation allows us to calculate the standard volume variation upon association and the dissociation constant at atmospheric pressure. On the other hand the native protein is undissociable at any pressure. In the simulative approach, the dissociation free energy has been calculated through the blue moon calculation method for the case of a multidimensional reaction coordinate, corrected for the rotational contribution within the semiclassical approximation for a free rigid-body rotor. The scheme permits to define a definite path for the rupture of the dimer and to calculate the effective force involved in the process. The calculated free energy difference is close to the experimental one, and the value obtained for the mutant is well below that obtained for the native protein, indicating that the theoretical reaction scheme is able to reproduce the experimental trend. Moreover, we find that, when the separation distance increases, the protein structure of the monomer is stable in line with the fast recovery of the original fluorescence properties after decompression, which excludes the presence of partly unfolded intermediates during the dimer-monomer transition.