STAT3 Mediates the Differential Effects of Oncostatin M and TNFα on RA Synovial Fibroblast and Endothelial Cell Function.

Objectives: Oncostatin M (OSM), a pleiotropic cytokine and a member of the gp130/IL-6 cytokine family, has been implicated in the pathogenesis of autoimmune diseases. Here we investigate the mechanisms by which its synergistic interactions with TNFα regulate the cellular bioenergetics and invasive function of synovial cells from patients with Rheumatoid Arthritis. Methods: Primary RA synovial fibroblasts (RAFLS) and human umbilical vein endothelial cells (HUVEC) were cultured with OSM alone or in combination with TNFα. Pro-inflammatory cytokines, angiogenic growth factors and adhesion molecules were quantified by real-time PCR and ELISA. Invasion, angiogenesis and cellular adhesion were quantified by Transwell invasion chambers, Matrigel tube formation assays, and adhesion binding assays. Cellular bioenergetics was assessed using the Seahorse XFe96 Analyser. Key metabolic genes (GLUT-1, HK2, PFKFB3, HIF1α, LDHA, PKM2) and transcription factor STAT3 were measured using real-time PCR and western blot. Results: OSM differentially regulates pro-inflammatory mediators in RAFLS and HUVEC, with IL-6, MCP-1, ICAM-1, and VEGF all significantly induced, in contrast to the observed inhibition of IL-8 and GROα, with opposing effects observed for VCAM-1 depending on cell type. Functionally, OSM significantly induced angiogenic network formation, adhesion, and invasive mechanisms. This was accompanied by a change in the cellular bioenergetic profile of the cells, where OSM significantly increased the ECAR/OCR ratio in favor of glycolysis, paralleled by induction of the glucose transporter GLUT-1 and key glycolytic enzymes (HK2, PFKFB3, HIF1α). OSM synergizes with TNFα to differentially regulate pro-inflammatory mechanisms in RAFLS and HUVEC. Interestingly, OSM differentially synergizes with TNFα to regulate metabolic reprogramming, where induction of glycolytic activity with concomitant attenuation of mitochondrial respiration and ATP activity was demonstrated in RAFLS but not in HUVEC. Finally, we identified a mechanism, whereby the combination of OSM with TNFα induces transcriptional activity of STAT3 only in RAFLS, with no effect observed in HUVEC. Conclusion: STAT3 mediates the differential effects of OSM and TNFα on RAFLS and EC function. Targeting OSM or downstream signaling pathways may lead to new potential therapeutic or adjuvant strategies, particularly for those patients who have sub-optimal responses to TNFi.

Induction of protein citrullination and auto-antibodies production in murine exposed to nickel nanomaterials.

Citrullination, or the post-translational deimination of polypeptide-bound arginine, is involved in several pathological processes in the body, including autoimmunity and tumorigenesis. Recent studies have shown that nanomaterials can trigger protein citrullination, which might constitute a common pathogenic link to disease development. Here we demonstrated auto-antibody production in serum of nanomaterials-treated mice. Citrullination-associated phenomena and PAD levels were found to be elevated in nanomaterials -treated cell lines as well as in the spleen, kidneys and lymph nodes of mice, suggesting a systemic response to nanomaterials injection, and validated in human pleural and pericardial malignant mesothelioma (MM) samples. The observed systemic responses in mice exposed to nanomaterials support the evidence linking exposure to environmental factors with the development of autoimmunity responses and reinforces the need for comprehensive safety screening of nanomaterials. Furthermore, these nanomaterials induce pathological processes that mimic those observed in Pleural MM, and therefore require further investigations into their carcinogenicity.

Semiconductor versus graphene quantum dots as fluorescent probes for cancer diagnosis and therapy applications.

Early diagnosis of cancer is of critical importance in determining the outcome of a patient, and nanoparticulate fluorophores have been at the centre of research for such applications owing to their superior optical properties. Furthermore, the large surface area to volume ratios of these fluorophores enables them to be endowed with several modalities, including the targeting of specific biomarkers and drug delivery capabilities, promoting them as therapeutic agents as well. Over the last few decades, semiconductor quantum dots have dominated the field due to their unique yet well characterised optical properties. However, the scope of their application for diagnosis and therapy of cancer has been hindered by declarations of in vivo toxicity attributed to heavy metals typically found in their composition. Recent arrivals graphene quantum dots, or carbon-derived counterparts to SQDs, are often claimed to be biocompatible but they have complicated optical properties. In this review, we compare the properties of these two types of quantum dots in view of their employment as fluorescent agents for cancer diagnosis and therapy.

Highly sensitive single domain antibody-quantum dot conjugates for detection of HER2 biomarker in lung and breast cancer cells.

Despite the widespread availability of immunohistochemical and other methodologies for screening and early detection of lung and breast cancer biomarkers, diagnosis of the early stage of cancers can be difficult and prone to error. The identification and validation of early biomarkers specific to lung and breast cancers, which would permit the development of more sensitive methods for detection of early disease onset, is urgently needed. In this paper, ultra-small and bright nanoprobes based on quantum dots (QDs) conjugated to single domain anti-HER2 (human epidermal growth factor receptor 2) antibodies (sdAbs) were applied for immunolabeling of breast and lung cancer cell lines, and their performance was compared to that of anti-HER2 monoclonal antibodies conjugated to conventional organic dyes Alexa Fluor 488 and Alexa Fluor 568. The sdAbs-QD conjugates achieved superior staining in a panel of lung cancer cell lines with differential HER2 expression. This shows their outstanding potential for the development of more sensitive assays for early detection of cancer biomarkers.

Heparin conjugated quantum dots for in vitro imaging applications.

In this work heparin-gelatine multi-layered cadmium telluride quantum dots (QDgel/hep) were synthesised using a novel 'one-pot' method. The QDs produced were characterised using various spectroscopic and physiochemical techniques. Suitable QDs were then selected and compared to thioglycolic acid stabilised quantum dots (QDTGA) and gelatine coated quantum dots (QDgel) for utilisation in in vitro imaging experiments on live and fixed permeabilised THP-1, A549 and Caco-2 cell lines. Exposure of live THP-1 cells to QDgel/hep resulted in localisation of the QDs to the nucleus of the cells. QDgel/hep show affinity for the nuclear compartment of fixed permeabilised THP-1 and A549 cells but remain confined to cytoplasm of fixed permeabilised Caco-2 cells. It is postulated that heparin binding to the CD11b receptor facilitates the internalisation of the QDs into the nucleus of THP-1 cells. In addition, the heparin layer may reduce the unfavourable thrombogenic nature of quantum dots observed in vivo. FROM THE CLINICAL EDITOR: In this study, heparin conjugated quantum dots were found to have superior imaging properties compared to its native counterparts. The authors postulate that heparin binding to the CD11b receptor facilitates QD internalization to the nucleus, and the heparin layer may reduce the in vivo thrombogenic properties of quantum dots.

Queuosine deficiency in eukaryotes compromises tyrosine production through increased tetrahydrobiopterin oxidation.

Queuosine is a modified pyrrolopyrimidine nucleoside found in the anticodon loop of transfer RNA acceptors for the amino acids tyrosine, asparagine, aspartic acid, and histidine. Because it is exclusively synthesized by bacteria, higher eukaryotes must salvage queuosine or its nucleobase queuine from food and the gut microflora. Previously, animals made deficient in queuine died within 18 days of withdrawing tyrosine, a nonessential amino acid, from the diet (Marks, T., and Farkas, W. R. (1997) Biochem. Biophys. Res. Commun. 230, 233-237). Here, we show that human HepG2 cells deficient in queuine and mice made deficient in queuosine-modified transfer RNA, by disruption of the tRNA guanine transglycosylase enzyme, are compromised in their ability to produce tyrosine from phenylalanine. This has similarities to the disease phenylketonuria, which arises from mutation in the enzyme phenylalanine hydroxylase or from a decrease in the supply of its cofactor tetrahydrobiopterin (BH4). Immunoblot and kinetic analysis of liver from tRNA guanine transglycosylase-deficient animals indicates normal expression and activity of phenylalanine hydroxylase. By contrast, BH4 levels are significantly decreased in the plasma, and both plasma and urine show a clear elevation in dihydrobiopterin, an oxidation product of BH4, despite normal activity of the salvage enzyme dihydrofolate reductase. Our data suggest that queuosine modification limits BH4 oxidation in vivo and thereby potentially impacts on numerous physiological processes in eukaryotes.

CdTe Quantum Dot/Dye Hybrid System as Photosensitizer for Photodynamic Therapy.

We have studied the photodynamic properties of novel CdTe quantum dots-methylene blue hybrid photosensitizer. Absorption spectroscopy, photoluminescence spectroscopy, and fluorescence lifetime imaging of this system reveal efficient charge transfer between nanocrystals and the methylene blue dye. Near-infrared photoluminescence measurements provide evidence for an increased efficiency of singlet oxygen production by the methylene blue dye. In vitro studies on the growth of HepG2 and HeLa cancerous cells were also performed, they point toward an improvement in the cell kill efficiency for the methylene blue-semiconductor nanocrystals hybrid system.

Photosensitizer methylene blue-semiconductor nanocrystals hybrid system for photodynamic therapy.

In this work we report on the development of novel hybrid material with enhanced photodynamic properties based on methylene blue and CdTe nanocrystals. Absorption spectroscopy, visible photoluminescence spectroscopy and fluorescence lifetime imaging of this system reveal efficient charge transfer between nanocrystals and the methylene blue dye. Near infra-red photoluminescence measurements provide evidence for an increased efficiency of singlet oxygen production by the methylene blue dye. In vitro studies on the growth of HepG2 and HeLa cancerous cells were also performed, they point towards an improvement in the cell kill efficiency for the methylene blue-semiconductor nanocrystals hybrid system.