Impaired mRNA Expression of the Migration Related Chemokine Receptor CXCR4 in Mesenchymal Stem Cells of COPD Patients.

Defective tissue repair and remodeling are main aspects of Chronic Obstructive Pulmonary Disease (COPD) pathophysiology. Bone marrow mesenchymal stem cells (BM-MSCs) have been implicated in this direction, as their functional impairment and recruitment could possibly contribute to disease development and progression. The present study characterizes for the first time the expression of migration related chemokine receptors and their ligands in BM-MSCs from COPD patients. CXCR4/SDF1a and CCR7/CCL19-CCL21 mRNA levels were evaluated in BM-MSCs obtained from twelve COPD patients and seven healthy donors. SDF1a protein levels in sera and BM-MSCs' conditioned media were also evaluated. CXCR4, SDF1a, CCL19, and CCL21 mRNA levels were significantly reduced in COPD BM-MSCs while CCR7 levels were undetectable. Notably, SDF1a protein levels were marginally elevated in both patient sera and BM-MSCs' conditioned media while the increase in SDF1a serum levels significantly correlated with disease severity in COPD. Our findings show posttranscriptional regulation of SDF1a levels in BM-MSCs of COPD patients and significant downregulation of SDF1a and CXCR4 mRNA indicating an involvement of the SDF1a signaling pathway in the disease pathophysiology.

Combined Pulmonary Fibrosis and Emphysema in Scleroderma-Related Lung Disease Has a Major Confounding Effect on Lung Physiology and Screening for Pulmonary Hypertension.

OBJECTIVE: To assess the prevalence of combined pulmonary fibrosis and emphysema (CPFE) in systemic sclerosis (SSc) patients with interstitial lung disease (ILD) and the effect of CPFE on the pulmonary function tests used to evaluate the severity of SSc-related ILD and the likelihood of pulmonary hypertension (PH). METHODS: High-resolution computed tomography (HRCT) scans were obtained in 333 patients with SSc-related ILD and were evaluated for the presence of emphysema and the extent of ILD. The effects of emphysema on the associations between pulmonary function variables and the extent of SSc-related ILD as visualized on HRCT and echocardiographic evidence of PH were quantified. RESULTS: Emphysema was present in 41 (12.3%) of the 333 patients with SSc-related ILD, in 26 (19.7%) of 132 smokers, and in 15 (7.5%) of 201 lifelong nonsmokers. When the extent of fibrosis was taken into account, emphysema was associated with significant additional differences from the expected values for diffusing capacity for carbon monoxide (DLco) (average reduction of 24.1%; P < 0.0005), and the forced vital capacity (FVC)/DLco ratio (average increase of 34.8%; P < 0.0005) but not FVC. These effects were identical in smokers and nonsmokers. Multivariate analysis showed that the presence of emphysema had a greater effect than echocardiographically determined PH on the FVC/DLco ratio, regardless of whether it was analyzed as a continuous variable or using a threshold value of 1.6 or 2.0. CONCLUSION: Among patients with SSc-related ILD, emphysema is sporadically present in nonsmokers and is associated with a low pack-year history in smokers. The confounding effect of CPFE on measures of gas exchange has major implications for the construction of screening algorithms for PH in patients with SSc-related ILD.

Increased bronchoalveolar lavage fluid CD1c expressing dendritic cells in idiopathic pulmonary fibrosis.

BACKGROUND: Chronic inflammation is implicated in idiopathic pulmonary fibrosis (IPF) pathogenesis and is associated with persistent activation of immune responses. These are largely controlled by dendritic cells (DCs). Although large numbers of DCs infiltrate the lungs of patients with IPF, there are no similar reports in bronchoalveolar lavage fluid (BALF). OBJECTIVES: We aimed to investigate DC populations in BALF of IPF patients. METHODS: CD1c(+) myeloid DCs, BDCA3(high) myeloid DCs, BDCA2(+) plasmacytoid DCs and CD83(+) mature DCs were identified by flow cytometry in the BALF of 10 IPF patients and 10 controls. DC numbers were expressed as percentages of total BALF leukocytes. RESULTS: CD1c(+) myeloid DCs were increased in IPF patients versus controls [median (ranges in parentheses) 1.16% (0.25-3.97) vs. 0.61% (0.19-1.10), p = 0.01]. There was also a trend towards increased BDCA3(high) myeloid DCs [0.57% (0.23-0.88) vs. 0.28% (0.07-0.96), p = 0.07]. No differences were reported in BDCA2(+) DCs and CD83(+) DCs between IPF patients and controls. CONCLUSIONS: IPF is associated with an increase in percentages of BALF myeloid DCs. Considering that such an increase was not observed in CD83(+) mature DCs, most of these DCs should be immature.

Synthesis of potent agonists of substance P by replacement of Met11 with Glu(OBzl) and N-terminal glutamine with Glp of the C-terminal hexapeptide and heptapeptide of substance P.

The analogues [Glp6,Glu(OBzl)11]SP(6-11) and [Glp5,Glu(OBzl)11]SP(5-11) of the C-terminal hexapeptide and heptapeptide of Substance P have been synthesized by conventional solution methods. In each analogue the N-terminal glutamine has been replaced by pyroglutamic acid, while the COOCH2C6H5 ester group has replaced the SCH3 group of the Met11 side chain. The in vitro activity of both analogues has been determined on three biological preparations: guinea pig ileum (GPI), rat vas deferens (RVD) and rat portal vein (RPV). The results showed that both analogues are highly potent and selective agonists on GPI through the NK-1 receptor. They are more potent than SP itself, with 1.54 and 1.25 respective values of relative potency on GPI. Their selectivity has been studied by utilizing atropine-treated guinea pig ileum (GPI+At). The analogues showed low activity on RVD and RPV tissues, which represent NK-2 and NK-3 monoreceptor assay, respectively.

Synthesis and biological activity of locust AKH-I and its analogues with modifications at the threonine residues.

A convenient method of synthesis, using a combination of solid and liquid phase methodology, for locust Adipokinetic Hormone-I (AKH-I) and its analogues with modifications at the threonine residues are reported. The N-terminal nonapeptide acid of AKH-I is synthesized in the solid phase using the 2-chlorotrityl chloride resin and the Fmoc/t-Bu strategy. Quantitative cleavage of the nonapeptide acid from the resin, with the tert-butyl type side-chain protection intact, is achieved with a mixture of acetic acid/trifluoroethanol/dichloromethane. The nonapeptide acid is then coupled in solution to the threonine derivatives, H-Thr-NH2 or H-Thr(Bzl)-NH2, with the DCC/HOBt method. The efficiency of this approach in the synthesis of AKH-I is demonstrated by the high yields and purity of the synthesized peptides. All the synthesized peptides were tested in two ways: first, in a lipid mobilization assay in locusts in vivo; and second, in a novel assay in vitro concerned with the uptake of radiolabelled acetate into locust tissue. Replacement of the hydroxyl hydrogen in Thr5 of locust AKH-I by the bulky and highly lipophilic tert-butyl group reduced the potency markedly, whereas efficacy is unaffected, but when the hydroxyl hydrogen of Thr10 in AKH-I is replaced by a benzyl group, the activity of the resulting analogue is identical to that of the natural peptide. Structure-activity relationships are discussed.

Synthesis of a potent antagonist of substance P by replacing the CH2SCH3 and the alpha-carboxamide groups of the methionine at [Orn6]-SP6-11 by benzyl ester groups.

Analogues of [Orn6]-SP6-11 have been synthesized in which the CH2SCH3 group of Met11 is replaced by a COOCH3 or a COOBzl group. These analogues, which were tested for agonist and antagonist activity in three in vitro preparations representative of NK-1, NK-2 and NK-3 receptor types, were full agonists at NK-1 receptors, showed very weak agonist activity at NK-2, receptors and were weak antagonists at NK-3 receptors. The above analogues were modified by substituting the alpha-carboxamide of residue 11 by a COOCH3 and a COOBzl group, respectively. The resulting analogues were found to be devoid of agonist activity in each of the functional assays. However, they showed weak antagonist activity at each receptor subtype, with the exception of the dibenzyl analogue, which was a potent and selective NK-1 receptor antagonist. It is concluded that appropriate modification of the side chain of Met11 and its alpha-carboxamide leads to a potent and selective at NK-1 receptor antagonist.

Synthesis of potent antagonists of substance P by modifying the methionyl and glutaminyl residues of its C-terminal hexapeptide and without using D-amino acids.

Analogues of [Orn6]-SP6-11 have been synthesized in which the Met11-NH2 residue is replaced by the alpha, gamma-dimethyl, alpha, gamma-dibenzyl and alpha, gamma-di-tert-butyl esters of glutamic acid. These analogues were tested in three in vitro preparations representative of NK-1, NK-2 and NK-3 receptor types for agonist and antagonist activity. The dimethyl analogue is a selective full agonist in the NK-1 receptor type and a weak antagonist in the other two receptor types, while the dibenzyl and the di-tert-butyl analogues are potent antagonists in the NK-1 receptor type and weak antagonists in the other two receptor types. It is concluded that appropriate modification at the alpha-carboxamide and the side chain of the methionine residue of substance P may induce antagonism without using D-amino acids.

Solid-phase synthesis and spectroscopic studies of TRH analogues incorporating cis- and trans-4-hydroxy-L-proline.

An efficient solid-phase synthesis of the TRH analogue Glp-His(Nim-Trt)-Hyp-OH is described. Na-Fmoc protected amino acids and DCC/HOBt activation were employed. The bulky and mild-acid-sensitive 2-chlorotrityl resin, utilised as the solid support, completely suppressed dioxopiperazine formation. The tripeptide is a key intermediate in the synthesis of TRH analogues incorporating cis- and trans-4-hydroxy-L-proline. The tripeptide was converted, with inversion of configuration at C-4 of the Hyp residue, to Glp-His(Nim-Trt)-cHyp lactone in the presence of triphenylphosphine-diethyl azodicarboxylate (TPP-DEAD). One-pot MeOH-TPP-DEAD transesterification of the lactone, followed by Nim-detritylation, provided Glp-His-cHyp-OMe. This ester gave the corresponding amide and acid on ammonolysis and saponification, respectively. A high-field 1H NMR investigation of Glp-His-cHyp-OH and its diastereomer Glp-His-Hyp-OH, obtained by Nim-detritylation of the key tripeptide, showed that the configuration at C-4 of the prolyl residues is critical for the determination of the preferred three-dimensional structure of the molecules.

Synthesis of a potent agonist of substance P by modifying the methionyl and glutaminyl residues of the C-terminal hexapeptide of substance P. Structure-activity relationships.

Analogues of [Orn6]-SP6-11 have been synthesized in which the Met11 residue is replaced by glutamate gamma-alkylesters. These analogues were tested in three in vitro preparations representative of NK-1, NK-2, and NK-3 receptor types. Substitution of the SCH3 group of the Met11 side chain by a COOR (R = methyl, ethyl, n-propyl, n-butyl, cyclohexyl) group results in analogues which are full agonists in NK-1 and NK-2 preparations but show little agonist activity in the NK-3 preparation. When the SCH3 group is replaced by a t-butyl ester group and the resulting analogue is a full agonist in all the above preparations and more active than the parent hexapeptide and SP-OCH3 at NK-1 receptors. It is concluded that for activity at NK-1 receptors methionine can be replaced by gamma-t-butyl glutamate without loss of activity, whilst at NK-2 and NK-3 receptors the above substitution increases the activity of [Orn6]-SP6-11. Other gamma-alkyl esters of the glutamic acid reduce its biological activity.

Synthesis and biological activity of substance P C-terminal hexapeptide and heptapeptide analogues.

The analogues [Glu(OBzl)11]SP6-11 and [Glu(OBzl)11]SP5-11 of the C-terminal hexapeptide and heptapeptide of Substance P have been synthesized by conventional solution methods. In each analogue the SCH3 group of Met11 is replaced by the COOCH2C6H5 group. The in vitro activity of both analogues has been determined on three biological preparations: guinea pig ileum (GPI), rat vas deferens (RVD), and rat portal vein (RPV). The selectivity for the different receptors has been studied by utilizing atropine-treated guinea pig ileum (GPI + At). The results showed that both analogues are mainly active on GPI through the NK-1 receptor and that both analogues are equipotent to Substance P.