Ewa Jankowska-Steifer

N-acetylcysteine inhibits IL-8 and MMP-9 release and ICAM-1 expression by bronchoalveolar cells from interstitial lung disease patients

N-acetylcysteine (NAC), owing to its antioxidant, mucolytic and anti-inflammatory properties, is used in the treatment of various pulmonary disorders. However, the direct effects of NAC on bronchoalveolar lavage (BAL) cells from patients suffering from interstitial lung diseases have not yet been studied. Therefore, the aim of the present work was to evaluate the effect of NAC on interleukin-8 (IL-8) and matrix metalloproteinase-9 (MMP-9) production as well as intercellular cell adhesion molecule-1 (ICAM-1) expression by BAL cells from interstitial lung diseases. The study was performed on BAL cells from nine patients with interstitial lung disease: four patients with idiopathic pulmonary fibrosis (IPF) and five patients with sarcoidosis. Cultured unstimulated BAL cells were treated with increasing doses of NAC (1-30 mM). Production of IL-8 and MMP-9 was evaluated by specific enzyme-linked immuno-sorbent assays and ICAM-1 expression was studied by immunohistochemistry. NAC exerted a dose-dependent inhibitory effect on IL-8 and MMP-9 release and ICAM- expression by BAL macrophages and lymphocytes from patients with IPF and sarcoidosis. In conclusion, NAC inhibits production of factors playing a key role in the etiopathogenesis of interstitial lung diseases, thus suggesting its possible therapeutic potency in the treatment of these disorders.

Fecal lactoferrin and Clostridium spp. in stools of autistic children

Stools from autistic and healthy children were studied for fecal lactoferrin, Clostridium difficile toxins, Clostridium perfringens enterotoxin and cultured for Clostridium spp. Elevated level of FLA was demonstrated in 24.4% stools, all from boys (31.25%). No toxins were detected. Clostridium spp. was isolated with similar frequency from all samples. C. perfringens were isolated significantly often from the autistic stools, intermediate sensitive strains to penicillin 19%, to clindamycin 11.3%, and to metronidazole 7.5% were detected. Further studies on fecal microflora and inflammatory mediators, with larger groups of patients, are required in order to explain their role in neurological deficits.

Cellular phenotypes and spatio-temporal patterns of lymphatic vessel development in embryonic mouse hearts

Background: The origin of cardiac lymphatics from venous endothelial cells or from scattered lymphangioblasts has been discussed in the literature. We aimed to establish the stage when lymphatic vessels appear in the developing mouse heart, the location of the first lymphatics, and to define cellular phenotypes of growing lymphatics. Results: We found that scattered Lyve‐1‐positive cells located in the subepicardial area of developing heart expressed CD45, CD68, F4/80, or CD11b but not CD31. Prox‐1+/Lyve‐1+ cellular cords or vessels were found to invade 12.5–13.5‐dpc hearts via two routes: from the venous pole, i.e., dorsal atrioventricular sulcus, or on the dorsal atrial surface from mediastinum and from the arterial pole, i.e., along the great arteries. The Prox‐1+/Lyve‐1+ vessels were located among the Prox‐1+/Lyve‐1− cords and among the scattered Prox‐1−/Lyve‐1+ cells. The Prox‐1+/Lyve‐1− cellular cords/tubules dominate initially at the arterial pole whereas Lyve‐1+/Prox‐1− cellular cords/tubules dominate initially on the venous pole, i.e., dorsal atrioventricular sulcus. The Lyve‐1+/CD45+, Lyve‐1+/CD11b+, Lyve‐1+/F4/80+ and Lyve‐1+/CD68+ cells were subsequently found to be co‐opted to the wall of the developing lymphatic vessels while gaining Flk‐1. Conclusions: Lymphatic primordia exhibit different cellular phenotypes and different spatiotemporal pattern on the venous pole as compared with the arterial pole of the heart. Developmental Dynamics 241:1473–1486, 2012.

3-D reconstruction and multiple marker analysis of mouse proepicardial endothelial cell population

BACKGROUND The proepicardium (PE), a transient embryonic structure crucial for the development of the epicardium and heart, contains its own population of endothelial cells (ECs). The aim of our study was to determine the pattern, anatomical orientation and phenotypic marker expression of the endothelial cell network within the PE. RESULTS Immunohistochemical findings revealed that proepicardial ECs express both early and late EC-specific markers such as CD31, Flk-1, Lyve-1 and Tie-2 but not SCL/Tal1, vWF, Dll4 or Notch1. Proepicardial ECs are present in the vicinity of the sinus venosus (SV) and form a continuous network of vascular sprouts/tubules connected with the SV endothelium, with Ter-119-positive erythroblasts in the vascular lumina. CONCLUSIONS On the basis of our results, we postulate the existence of a continuous network of ECs in the PE, exhibiting connection and/or patency with the SV and forming vessels/tubules/strands. Marker expression suggests that ECs are immature and undifferentiated, which was also confirmed with a transmission electron microscopy (TEM) analysis. Our results deliver new data for a better understanding of the nature of proepicardial ECs.

SK053 triggers tumor cells apoptosis by oxidative stress-mediated endoplasmic reticulum stress

Thioredoxins (Trx) together with thioredoxin reductases (TrxR) participate in the maintenance of protein thiol homeostasis and play cytoprotective roles in tumor cells. Therefore, thioredoxin-thioredoxin reductase system is considered to be a promising therapeutic target in cancer treatment. We have previously reported that SK053, a peptidomimetic compound targeting the thioredoxin-thioredoxin reductase system, induces oxidative stress and demonstrates antitumor activity in mice. In this study, we investigated the mechanisms of SK053-mediated tumor cell death. Our results indicate that SK053 induces apoptosis of Raji cells accompanied by the activation of the endoplasmic reticulum (ER) stress and induction of unfolded protein response. Incubation of tumor cells with SK053 induces increase in BiP, CHOP, and spliced XBP-1 levels, which precede induction of apoptosis. CHOP-deficient (CHOP(-/-)) mouse embryonic fibroblasts are more resistant to SK053-induced apoptosis as compared with normal fibroblasts indicating that the apoptosis of tumor cells depends on the expression of this transcription factor. Additionally, the ER-stress-induced apoptosis, caused by SK053, is strongly related with Trx expression levels. Altogether, our results indicate that SK053 induces ER stress-associated apoptosis and reveal a link between thioredoxin inhibition and induction of UPR in tumor cells.

Cardiac mouse lymphatics: developmental and anatomical update.

The adult mouse heart possesses an extensive lymphatic plexus draining predominantly the subepicardium and the outer layer of the myocardial wall. However, the development of this plexus has not been entirely explored, partially because of the lack of suitable methods for its visualization as well as prolonged lymphatic vessel formation that starts prenatally and proceeds during postnatal stages. Also, neither the course nor location of collecting vessels draining lymph from the mouse heart have been precisely characterized. In this article, we report that murine cardiac lymphatic plexus development that is limited prenatally only to the subepicardial area, postnatally proceeds from the subepicardium toward the myocardial wall with the base‐to‐apex gradient; this plexus eventually reaches the outer half of the myocardium with a predominant location around branches of coronary arteries and veins. Based on multiple marker immunostaining, the molecular marker‐phenotype of cardiac lymphatic endothelial cells can be characterized as: Prox‐1+, Lyve‐1+, VEGFR3+, Podoplanin+, VEGFR2+, CD144+, Tie2+, CD31+, vWF−, CD34−, CD133−. There are two major collecting vessels: one draining the right and left ventricles along the left conal vein and running upwards to the left side of the pulmonary trunk and further to the nearest lymph nodes (under the aortic arch and near the trachea), and the other one with its major branch running along the left cardiac vein and further on the surface of the coronary sinus and the left atrium to paratracheal lymph nodes. The extracardiac collectors gain the smooth muscle cell layer during late postnatal stages. Anat Rec, 297:1115–1130, 2014.

Keratinization of outer root sheath cells is prevented by contact with inner root sheath of rat hair follicles

The purpose of the present study was to elucidate why keratinocytes of the outer root sheath (ORS) do not keratinize in situ. Two possibilities were considered—inhibition of keratinization is caused by contact of ORS with inner root sheath (IRS) or insufficient supply of keratinization promoting factors from the surrounding tissues to the ORS. In order to distinguish between these possibilities mid-segments of hair follicles were liberated from the dermis by dissection followed by collagenase digestion. ORS cells were then either allowed to migrate from the mid-segments or were kept on the agarose layer which prevented cell spreading and preserved three dimensional structure of hair root. Cultures were stimulated with calcium or EGF, and studied morphologically at the light and transmission electron microscope level. The level of mRNA for differentiation cell markers was also studied by RealTime PCR. ORS cells growing in a medium with low Ca2+ content formed monolayers, which after elevation of Ca2+ produced multilayers with cells containing keratohyalin-like granules. Ca2+ or EGF treatment upregulated expression of involucrin, filaggrin and keratinocyte differentiation associated protein (Kdap). Culture of mid-segments of hair follicles in low calcium culture medium kept on agarose increased expression of filaggrin and Kdap, but downregulated expression of involucrin. Stimulation by Ca2+ further increased expression of filaggrin and Kdap, but had no effect on the level of involucrin expression. EGF stimulated expression of filaggrin only. It is concluded that IRS exerted an inhibitory effect on the expression of involucrin, an essential component of the cornified envelope, thus preventing keratinization of ORS cells in situ. On the other hand, improved access of nutrients or promoting factors of keratinization to the mid-segment of hair follicles augmented expression of filaggrin and Kdap, proteins engaged in the differentiation of keratinocytes but not involved in its terminal phase.

Mouse Proepicardium Exhibits a Sprouting Response to Exogenous Proangiogenic Growth Factors in vitro.

Angiogenesis contributes to the generation of the vascular bed but also affects the progression of many diseases, such as tumor growth. Many details of the molecular pathways controlling angiogenesis are still undefined due to the lack of appropriate models. We propose the proepicardial explant as a suitable model for studying certain aspects of angiogenesis. The proepicardium (PE) is a transient embryonic structure that contains a population of undifferentiated endothelial cells (ECs) forming a vascular net continuous with the sinus venosus. In this paper, we show that PE explants give rise to CD31-positive vascular sprouts in the presence of basic fibroblast growth factor (bFGF) and 2 isoforms of vascular endothelial growth factor A (VEGF-A), i.e. VEGF-A120 and VEGF-A164. Vascular sprouts exhibit differences in number, length, thickness and the number of branches, depending on the combination of growth factors used. Moreover, the ECs of the sprouts express various levels of mRNA for Notch1 and its ligand Dll4. Additionally, stimulation with bFGF/VEGF-A164 upregulates the expression of Lyve-1 antigen in the ECs in the sprouts. In summary, we present a new model for angiogenesis studies involving mouse PE as a source of ECs. We believe that our model may act as a supplementary assay for angiogenesis studies along with the existing models.

Hydrostatic and boundary lubrication of joints--nature of boundary lubricant.

A very low coefficient of friction in joints makes it difficult to define clearly the mechanism of cartilage lubrication. The present paper describes the two currently predominant and mutually complementary views aiming to elucidate this mechanism. The first mechanism, referred to as hydrostatic lubrication, involves interstitial fluid pressurization from the cartilage and its importance for the formation of a layer separating the weight-bearing surfaces. The second mechanism, called boundary lubrication, assumes the existence of a substance that binds to the cartilage surface, permanently separating the friction elements. It has not been clearly determined which substances occurring in the synovial fluid function as boundary lubricants. The authors briefly describe the physicochemical properties of lubricin, surface-active phospholipids and hyaluronic acid, including their role in boundary lubrication.

Dominant ELOVL1 mutation causes neurological disorder with ichthyotic keratoderma, spasticity, hypomyelination and dysmorphic features

Background Ichthyosis and neurological involvement occur in relatively few known Mendelian disorders caused by mutations in genes relevant both for epidermis and neural function. Objectives To identify the cause of a similar phenotype of ichthyotic keratoderma, spasticity, mild hypomyelination (on MRI) and dysmorphic features (IKSHD) observed in two unrelated paediatric probands without family history of disease. Methods Whole exome sequencing was performed in both patients. The functional effect of prioritised variant in ELOVL1 (very-long-chain fatty acids (VLCFAs) elongase) was analysed by VLCFA profiling by gas chromatography–mass spectrometry in stably transfected HEK2932 cells and in cultured patient’s fibroblasts. Results Probands shared novel heterozygous ELOVL1 p.Ser165Phe mutation (de novo in one family, while in the other family, father could not be tested). In transfected cells p.Ser165Phe: (1) reduced levels of FAs C24:0-C28:0 and C26:1 with the most pronounced effect for C26:0 (P=7.8×10−6 vs HEK293 cells with wild type (wt) construct, no difference vs naïve HEK293) and (2) increased levels of C20:0 and C22:0 (P=6.3×10−7, P=1.2×10−5, for C20:0 and C22:0, respectively, comparison vs HEK293 cells with wt construct; P=2.2×10−7, P=1.9×10−4, respectively, comparison vs naïve HEK293). In skin fibroblasts, there was decrease of C26:1 (P=0.014), C28:0 (P=0.001) and increase of C20:0 (P=0.033) in the patient versus controls. There was a strong correlation (r=0.92, P=0.008) between the FAs profile of patient’s fibroblasts and that of p.Ser165Phe transfected HEK293 cells. Serum levels of C20:0–C26:0 FAs were normal, but the C24:0/C22:0 ratio was decreased. Conclusion The ELOVL1 p.Ser165Phe mutation is a likely cause of IKSHD.