Human blood-labyrinth barrier model to study the effects of cytokines and inflammation

Hearing loss is one of the 10 leading causes of disability worldwide. No drug therapies are currently available to protect or restore hearing. Inner ear auditory hair cells and the blood-labyrinth barrier (BLB) are critical for normal hearing, and the BLB between the systemic circulation and stria vascularis is crucial for maintaining cochlear and vestibular homeostasis. BLB defects are associated with inner ear diseases that lead to hearing loss, including vascular malformations, inflammation, and Meniere’s disease (MD). Antibodies against proteins in the inner ear and cytokines in the cochlea, including IL-1α, TNF-α, and NF-kβ, are detected in the blood of more than half of MD patients. There is also emerging evidence of inner ear inflammation in some diseases, including MD, progressive sensorineural hearing loss, otosclerosis, and sudden deafness. Here, we examined the effects of TNF-α, IL6, and LPS on human stria vascularis-derived primary endothelial cells cultured together with pericytes in a Transwell system. By measuring trans-endothelial electrical resistance, we found that TNF-α causes the most significant disruption of the endothelial barrier. IL6 had a moderate influence on the barrier, whereas LPS had a minimal impact on barrier integrity. The prominent effect of TNF-α on the barrier was confirmed in the expression of the major junctional genes responsible for forming the tight endothelial monolayer, the decreased expression of ZO1 and OCL. We further tested permeability using 2 μg of daptomycin (1,619 Da), which does not pass the BLB under normal conditions, by measuring its passage through the barrier by HPLC. Treatment with TNF-α resulted in higher permeability in treated samples compared to controls. LPS-treated cells behaved similarly to the untreated cells and did not show differences in permeability compared to control. The endothelial damage caused by TNF-α was confirmed by decreased expression of an essential endothelial proteoglycan, syndecan1. These results allowed us to create an inflammatory environment model that increased BLB permeability in culture and mimicked an inflammatory state within the stria vascularis

Detection of Ru potential metallodrug in human urine by MALDI-TOF mass spectrometry: validation and options to enhance the sensitivity

We studied the possibility of detection of [Ru(η5 -C5H5)(PPh3)2Cl] (abbreviated by RuCp) complex as a model system for Ru-based metallodrugs in human urine by using matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOF MS) without previous purification or removal of inorganic salts. Inorganic salts might prevent the detection of RuCp by MALDI-TOF MS, most likely through the increased number and intensity of background/organic matrix signals. This problem might be overcome by the acquisition of matrix free spectra and the addition of nanoparticles, such as carbon dots, to the urine solution. Our results suggest that RuCp is easily detectable by MALDI-TOF MS in all acquisition conditions, with the CHCA matrix being the best for acquisition in phosphate-containing solutions, whereas in urine, DHB and matrix-free approach demonstrated the highest sensitivity, precision, and reproducibility. The sensitivity of matrix-free MALDI detection of RuCp could be increased by the addition of carbon dots to the urine. Based on theoretical calcu lations for all matrix/analyte combinations, the model for the interaction of RuCp with carbon dots was established, and higher sensitivity explained.info:eu-repo/semantics/publishedVersio

Lack of NHE6 and Inhibition of NKCC1 Associated With Increased Permeability in Blood Labyrinth Barrier-Derived Endothelial Cell Layer.

Acoustic trauma, autoimmune inner ear disease, and presbycusis feature loss of the integrity of the blood-labyrinth barrier (BLB). Normal BLB function depends on endothelial structural integrity, which is supported and maintained by tight junctions and adherens junctions within the microvascular endothelial layer. When these junctions are disrupted, vascular leakage occurs. Tight junctions and adherens junctions are functionally and structurally linked, but the exact signaling pathways underlying their interaction remain unknown. In addition, solute carriers (SC) are essential for optimal exchange through BLB. Previously, we found that SC family member, the sodium-hydrogen exchanger NHE6, was expressed in all wildtype cochlear tissues, and that Nhe6-knockout mice displayed moderate hearing loss. Moreover, NHE6 depletion affected Trk protein turnover and endosomal signaling. Here, we investigated whether NHE6 might impact BLB integrity. We found that Nhe6-knockout, BLB-derived endothelial cells showed reduced expression of major junctional genes: Tjp1, F11r, Ocln, Cdh5, and Cldn5. Co-culturing BLB-derived endothelial cells with pericytes and/or perivascular resident macrophage-like melanocytes in a transwell system showed that monolayers of Nhe6-knockout BLB-derived cells had lower electrical resistance and higher permeability, compared to wildtype endothelial monolayers. Additionally, another SC, NKCC1, which was previously linked to congenital deafness, was downregulated in our Nhe6-knockout mouse model. Blocking NKCC1 with a NKCC1-specific inhibitor, bumetanide, in wildtype BLB-derived endothelial cells also caused the downregulation of major junctional proteins, particularly Tjp1 and F11r, which encode the zonula occludens and junctional adhesion molecule-1 proteins, respectively. Moreover, bumetanide treatment increased cell permeability. In conclusion, we showed that the lack or inhibition of NHE6 or NKCC1 affected the permeability of endothelial BLB-derived cells. These findings suggested that NHE6 and NKCC1 could serve as potential targets for modifying BLB permeability to facilitate drug delivery across the BLB to the cochlea or to protect the cochlea from ototoxic insults

Sodium-hydrogen exchanger 6 (NHE6) deficiency leads to hearing loss, via reduced endosomal signalling through the BDNF/Trk pathway.

Acid-base homeostasis is critical for normal growth, development, and hearing function. The sodium-hydrogen exchanger 6 (NHE6), a protein mainly expressed in early and recycling endosomes, plays an important role in regulating organellar pH. Mutations in NHE6 cause complex, slowly progressive neurodegeneration. Little is known about NHE6 function in the mouse cochlea. Here, we found that all NHE isoforms were expressed in wild-type (WT) mouse cochlea. Nhe6 knockout (KO) mice showed significant hearing loss compared to WT littermates. Immunohistochemistry in WT mouse cochlea showed that Nhe6 was localized in the organ of Corti (OC), spiral ganglion (SG), stria vascularis (SV), and afferent nerve fibres. The middle and the inner ears of WT and Nhe6 KO mice were not different morphologically. Given the putative role of NHE6 in early endosomal function, we examined Rab GTPase expression in early and late endosomes. We found no change in Rab5, significantly lower Rab7, and higher Rab11 levels in the Nhe6 KO OC, compared to WT littermates. Because Rabs mediate TrkB endosomal signalling, we evaluated TrkB phosphorylation in the OCs of both strains. Nhe6 KO mice showed significant reductions in TrkB and Akt phosphorylation in the OC. In addition, we examined genes used as markers of SG type I (Slc17a7, Calb1, Pou4f1, Cal2) and type II neurons (Prph, Plk5, Cacna1g). We found that all marker gene expression levels were significantly elevated in the SG of Nhe6 KO mice, compared to WT littermates. Anti-neurofilament factor staining showed axon loss in the cochlear nerves of Nhe6 KO mice compared to WT mice. These findings indicated that BDNF/TrkB signalling was disrupted in the OC of Nhe6 KO mice, probably due to TrkB reduction, caused by over acidification in the absence of NHE6. Thus, our findings demonstrated that NHEs play important roles in normal hearing in the mammalian cochlea

Analytical Platforms for the Determination of Phospholipid Turnover in Breast Cancer Tissue: Role of Phospholipase Activity in Breast Cancer Development

Altered lipid metabolism has been associated with the progression of various cancers, and aberrant expression of enzymes involved in the lipid metabolism has been detected in different stages of cancer. Breast cancer (BC) is one of the cancer types known to be associated with alterations in the lipid metabolism and overexpression of enzymes involved in this metabolism. It has been demonstrated that inhibition of the activity of certain enzymes, such as that of phospholipase A2 in BC cell lines sensitizes these cells and decreases the IC50 values for forthcoming therapy with traditional drugs, such as doxorubicin and tamoxifen. Moreover, other phospholipases, such as phospholipase C and D, are involved in intracellular signal transduction, which emphasizes their importance in cancer development. Finally, BC is assumed to be dependent on the diet and the composition of lipids in nutrients. Despite their importance, analytical approaches that can associate the activity of phospholipases with changes in the lipid composition and distribution in cancer tissues are not yet standardized. In this review, an overview of various analytical platforms that are applied on the study of lipids and phospholipase activity in BC tissues will be given, as well as their association with cancer diagnosis and tumor progression. The methods that are applied to tissues obtained from the BC patients will be emphasized and critically evaluated, regarding their applicability in oncology

Inhibitory effect of cisplatin and [Pt(dach)Cl2] on the activity of phospholipase A2

This work has been focused on testing the influence of two selected Pt(II) complexes cisplatin, Pt(NH3)2Cl2, and [Pt(dach)Cl2] on the activity of porcine pancreatic phospholipase A2 (PLA2). It has been assumed that this enzyme plays a role in carcinogenesis and that it could be a target in the tumour therapy. The results of this study show that both Pt(II) complexes inhibit the activity of the enzyme, though they bind to it in a different manner. While cisplatin interacts with the enzyme in an acompetitive manner, the stable interaction of [Pt(dach)Cl2] with PLA2 could not be detected under our experimental conditions. © 2013 Informa UK, Ltd

Comparison of MALDI-TOF mass spectra of [PdCl(dien)]Cl and [Ru(en) <inf>2</inf>Cl <inf>2</inf>]Cl acquired with different matrices

In this work, the matrix-assisted laser desorption and ionization time- -of-flight (MALDI-TOF) mass spectra of two cationic complexes, i.e., [PdCl(dien)]Cl and [Ru(en) 2Cl 2]Cl, acquired under different conditions were analyzed. The spectra were recorded with three matrices with or without trifluoroacetic acid (TFA), i.e., two traditional matrices, i.e., 2,5-dihydroxybenzoic acid and α-cyano-hydroxycinnamic acid, and one flavonoid, quercetin. The spectra acquired with quercetin appeared to be the simplest, whereas in the spectra obtained with other matrices, peaks arising either from the addition of matrix molecules or from the fragmentation products were detectable. Addition of TFA did not complicate the spectra of the Pd(II) and Ru(III) complexes when the traditional matrices were used. On the other hand, the spectra of Pd complex were simpler, whereas the addition of TFA in the case of the Ru complex resulted in a higher number of peaks, some of which could not be identified. Taken together, the results of this study once more emphasize the differences arising in the MALDI-TOF mass spectra of transition metal complexes in dependence on the applied matrix. Copyright 2011 (CC) SCS

Investigations of the lysophospholipid composition of human neutrophils under different stimulation conditions by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) is usually used for the analyses of proteins, carbohydrates and oligonucleotides. In spite of the number of advantages that MALDI-TOF MS exhibits for lipid analysis, this method has not often been applied in this field. In this paper we have extended our previous studies on the suitability of MALDI-TOF MS for the investigation of changes in the content of lipid-derived second messengers in organic extracts of human neutrophils. Qualitative differences in the lysophospholipid composition in organic extracts of the human neutrophils under different stimulation conditions could be easily observed by MALDI-TOF MS. Although there are still some methodological problems to be solved before this method can be routinely applied for the quantification of different lipid classes in complex biological mixtures (such as organic extracts of human neutrophils) it is shown here that MALDI-TOF MS possesses the capability to be used as a simple screening method for the investigation of the content of lipid-derived second messengers and of signalling pathways in cells

Image_1_Human blood-labyrinth barrier model to study the effects of cytokines and inflammation.JPEG

Hearing loss is one of the 10 leading causes of disability worldwide. No drug therapies are currently available to protect or restore hearing. Inner ear auditory hair cells and the blood-labyrinth barrier (BLB) are critical for normal hearing, and the BLB between the systemic circulation and stria vascularis is crucial for maintaining cochlear and vestibular homeostasis. BLB defects are associated with inner ear diseases that lead to hearing loss, including vascular malformations, inflammation, and Meniere’s disease (MD). Antibodies against proteins in the inner ear and cytokines in the cochlea, including IL-1α, TNF-α, and NF-kβ, are detected in the blood of more than half of MD patients. There is also emerging evidence of inner ear inflammation in some diseases, including MD, progressive sensorineural hearing loss, otosclerosis, and sudden deafness. Here, we examined the effects of TNF-α, IL6, and LPS on human stria vascularis-derived primary endothelial cells cultured together with pericytes in a Transwell system. By measuring trans-endothelial electrical resistance, we found that TNF-α causes the most significant disruption of the endothelial barrier. IL6 had a moderate influence on the barrier, whereas LPS had a minimal impact on barrier integrity. The prominent effect of TNF-α on the barrier was confirmed in the expression of the major junctional genes responsible for forming the tight endothelial monolayer, the decreased expression of ZO1 and OCL. We further tested permeability using 2 μg of daptomycin (1,619 Da), which does not pass the BLB under normal conditions, by measuring its passage through the barrier by HPLC. Treatment with TNF-α resulted in higher permeability in treated samples compared to controls. LPS-treated cells behaved similarly to the untreated cells and did not show differences in permeability compared to control. The endothelial damage caused by TNF-α was confirmed by decreased expression of an essential endothelial proteoglycan, syndecan1. These results allowed us to create an inflammatory environment model that increased BLB permeability in culture and mimicked an inflammatory state within the stria vascularis.</p