Photobiomodulation of oral fibroblasts stimulated with periodontal pathogens

Photobiomodulation (PBM) utilises light energy to treat oral disease, periodontitis. However, there remains inconsistency in the reporting of treatment parameters and a lack of knowledge as to how PBM elicits its molecular effects in vitro. Therefore, this study aimed to establish the potential immunomodulatory effects of blue and near infra-red light irradiation on gingival fibroblasts (GFs), a key cell involved in the pathogenesis of periodontitis. GFs were seeded in 96-well plates in media + / − Escherichia coli lipopolysaccharide (LPS 1 μg/ml), or heat-killed Fusobacterium nucleatum (F. nucleatum, 100:1MOI) or Porphyromonas gingivalis (P. gingivalis, 500:1MOI). Cultures were incubated overnight and subsequently irradiated using a bespoke radiometrically calibrated LED array (400–830 nm, irradiance: 24 mW/cm2 dose: 5.76 J/cm2). Effects of PBM on mitochondrial activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and adenosine triphosphate (ATP) assays, total reactive oxygen species production (ROS assay) and pro-inflammatory/cytokine response (interleukin-8 (IL-8) and tumour growth factor-β1 (TGFβ1)) were assessed 24 h post-irradiation. Data were analysed using one-way ANOVA followed by the Tukey test. Irradiation of untreated (no inflammatory stimulus) cultures at 400 nm induced 15%, 27% and 13% increases in MTT, ROS and IL-8 levels, respectively (p < 0.05). Exposure with 450 nm light following application of P. gingivalis, F. nucleatum or LPS induced significant decreases in TGFβ1 secretion relative to their bacterially stimulated controls (p < 0.001). Following stimulation with P. gingivalis, 400 nm irradiation induced 14% increases in MTT, respectively, relative to bacteria-stimulated controls (p < 0.05). These findings could identify important irradiation parameters to enable management of the hyper-inflammatory response characteristic of periodontitis.

Vascular endothelial growth factor-mediated peritoneal neoangiogenesis in peritoneal dialysis

Peritoneal dialysis (PD) is an important renal replacement therapy for patients with end-stage renal diseases, which is limited by peritoneal neoangiogenesis leading to ultrafiltration failure (UFF). Vascular endothelial growth factor (VEGF) and its receptors are key angiogenic factors involved in almost every step of peritoneal neoangiogenesis. Impaired mesothelial cells are the major sources of VEGF in the peritoneum. The expression of VEGF will be up-regulated in specific pathological conditions in PD patients, such as with non-biocompatible peritoneal dialysate, uremia and inflammation, and so on. Other working cells (i.e. vascular endothelial cells, macrophages and adipocytes) can also stimulate the secretion of VEGF. Meanwhile, hypoxia and activation of complement system further aggravate peritoneal injury and contribute to neoangiogenesis. There are several signalling pathways participating in VEGF-mediated peritoneal neoangiogenesis including tumour growth factor-β, Wnt/β-catenin, Notch and interleukin-6/signal transducer and activator of transcription 3. Moreover, VEGF is highly expressed in dialysate effluent of long-term PD patients and is associated with peritoneal transport function, which supports its role in the alteration of peritoneal structure and function. In this review, we systematically summarize the angiogenic effect of VEGF and evaluate it as a potential target for the prevention of peritoneal neoangiogenesis and UFF. Preservation of the peritoneal membrane using targeted therapy of VEGF-mediated peritoneal neoangiogenesis may increase the longevity of the PD modality for those who require life-long dialysis.

Dermatopontin, A Novel Adipokine Promoting Adipose Tissue Extracellular Matrix Remodelling and Inflammation in Obesity

Compelling evidence suggests that dermatopontin (DPT) regulates collagen and fibronectin fibril formation, the induction of cell adhesion and the prompting of wound healing. We aimed to evaluate the role of DPT on obesity and its associated metabolic alterations as well as its impact in visceral adipose tissue (VAT) inflammation and extracellular matrix (ECM) remodelling. Samples obtained from 54 subjects were used in a case-control study. Circulating and VAT expression levels of DPT as well as key ECM remodelling- and inflammation-related genes were analysed. The effect of pro- and anti-inflammatory mediators on the transcript levels of DPT in visceral adipocytes was explored. The impact of DPT on ECM remodelling and inflammation pathways was also evaluated in cultured adipocytes. We show that obesity and obesity-associated type 2 diabetes (T2D) increased (p < 0.05) circulating levels of DPT. In this line, DPT mRNA in VAT was increased (p < 0.05) in obese patients with and without T2D. Gene expression levels of DPT were enhanced (p < 0.05) in human visceral adipocytes after the treatment with lipopolysaccharide, tumour growth factor (TGF)- and palmitic acid, whereas a downregulation (p < 0.05) was detected after the stimulation with interleukin (IL)-4 and IL-13, critical cytokines mediating anti-inflammatory pathways. Additionally, we revealed that DPT increased (p < 0.05) the expression of ECM- (COL6A3, ELN, MMP9, TNMD) and inflammation-related factors (IL6, IL8, TNF) in human visceral adipocytes. These findings provide, for the first time, evidence of a novel role of DPT in obesity and its associated comorbidities by influencing AT remodelling and inflammation.

Scleroderma-specific autoantibodies embedded in immune complexes mediate endothelial damage: an early event in the pathogenesis of systemic sclerosis

Background: Consistently with their diagnostic and prognostic value, autoantibodies specific for systemic sclerosis (SSc) embedded in immune complexes (ICs) elicited a pro-inflammatory and pro-fibrotic cascade in healthy skin fibroblasts, engaging Toll-like Receptors (TLRs) via their nucleic acid components. The objective of this study was to investigate the pathogenicity of SSc-ICs in endothelial cells (ECs).Methods: ICs were purified from sera of SSc patients bearing different autoantibody specificities (antibodies against DNA topoisomerase I, centromeric proteins, RNA polymerase and Th/To), patients with systemic lupus erythematosus (SLE), primary anti-phospholipid syndrome (PAPS) or healthy controls (NHS) using polyethylen glycol precipitation. Human umbilical vein ECs (HUVECs) were incubated with ICs, IL-1β, Poly I:C, LPS or ODN CpG. mRNA levels of endothelin-1 (ET-1), collagenIα1 (colIα1), interferon (IFN)-α and IFN-β were investigated by Real-Time PCR; ICAM-1 expression was evaluated by cell-ELISA; secretion of IL-6, IL-8 and tumour growth factor (TGF)-β1 in culture supernatants was measured by ELISA. Intracellular signalling pathways culminating with NFkB, p38MAPK, SAPK-JNK and Akt were assessed by Western Blotting. Healthy skin fibroblasts were stimulated with supernatants from HUVEC incubated with ICs, and tumour growth factor (TGF)-β1secretion, mRNA levels of colIα1 and matrix metalloproteinase (mmp)-1 were evaluated.Results: All SSc stimulated IL-6 secretion compared to NHS-ICs; ACA-ICs and anti-Th/To-ICs increased ICAM-1 expression; all SSc-ICs but anti-Th/To-ICs augmented IL-8 levels; all SSc-ICs but ACA and ARA-ICs up-regulated et-1 and all SSc-ICs but ARA-ICs affected TGF-β1 secretion. ColIα1, IFN-α and IFN-β mRNA levels were not affected by any SSc-ICs. A differential modulation of tlr expression was observed: tlr2, tlr3 and tlr4 were upregulated by ATA-ICs and ACA-ICs, while anti-Th/To-ICs resulted in tlr9 up-regulation. All SSc-ICs activated p38MAPK and AKT, all SSc-ICs but ARA-ICs yielded the activation of NFκB; ATA-ICs and ACA-ICs increased the activation rate of both subunits of SAPK-JNK.When healthy skin fibroblasts were stimulated with supernatants from HUVECs incubated with SSc-ICs, TGF-β1 secretion and colIα1 expression levels were significantly modulated.Conclusions: These data provide the first demonstration of the pathogenicity of ICs from scleroderma patients with different autoantibodies in ECs. Endothelial activation induced by SSc-ICs ultimately led to a pro-fibrotic phenotype in healthy skin fibroblasts.

Co-factor-independent phosphoglycerate mutase of Leishmania donovani modulates macrophage signalling and promotes T-cell repertoires bearing epitopes for both MHC-I and MHC-II

SUMMARYImmunoactivation depends upon the antigen potential to modulate T-cell repertoires. The present study has enumerated the effect of 61 kDa recombinant Leishmania donovani co-factor-independent phosphoglycerate mutase (rLd-iPGAM) on mononuclear cells of healthy and treated visceral leishmaniasis subjects as well as on THP-1 cell line. rLd-iPGAM stimulation induced higher expression of interleukin-1β (IL-1β) in the phagocytic cell, its receptor and CD69 on T-cell subsets. These cellular activations resulted in upregulation of host-protective cytokines IL-2, IL-12, IL-17, tumour necrosis factor-α and interferon-γ, and downregulation of IL-4, IL-10 and tumour growth factor-β. This immune polarization was also evidenced by upregulation of nuclear factor-κ light-chain enhancer of activated B cells p50 and regulated expression of suppressor of mother against decapentaplegic protein-4. rLd-iPGAM stimulation also promoted lymphocyte proliferation and boosted the leishmaniacidal activity of macrophages by upregulating reactive oxygen species. It also induced 1·8-fold higher release of nitric oxide (NO) by promoting the transcription of inducible nitric oxide synthase gene. Besides, in silico analysis suggested the presence of major histocompatibility complex class I and II restricted epitopes, which can proficiently trigger CD8+ and CD4+ cells, respectively. This study reports rLd-iPGAM as an effective immunoprophylactic agent, which can be used in future vaccine design.

A miRNA signature associated with human metastatic medullary thyroid carcinoma

MicroRNAs (miRNAs) represent a class of small, non-coding RNAs that control gene expression by targeting mRNA and triggering either translational repression or RNA degradation. The objective of our study was to evaluate the involvement of miRNAs in human medullary thyroid carcinoma (MTC) and to identify the markers of metastatic cells and aggressive tumour behaviour. Using matched primary and metastatic tumour samples, we identified a subset of miRNAs aberrantly regulated in metastatic MTC. Deregulated miRNAs were confirmed by quantitative real-time PCR and validated by in situ hybridisation on a large independent set of primary and metastatic MTC samples. Our results uncovered ten miRNAs that were significantly expressed and deregulated in metastatic tumours: miR-10a, miR-200b/-200c, miR-7 and miR-29c were down-regulated and miR-130a, miR-138, miR-193a-3p, miR-373 and miR-498 were up-regulated. Bioinformatic approaches revealed potential miRNA targets and signals involved in metastatic MTC pathways. Migration, proliferation and invasion assays were performed in cell lines treated with miR-200 antagomirs to ascertain a direct role for this miRNA in MTC tumourigenesis. We show that the members of miR-200 family regulate the expression of E-cadherin by directly targeting ZEB1 and ZEB2 mRNA and through the enhanced expression of tumour growth factor β (TGFβ)-2 and TGFβ-1. Overall, the treated cells shifted to a mesenchymal phenotype, thereby acquiring an aggressive phenotype with increased motility and invasion. Our data identify a robust miRNA signature associated with metastatic MTC and distinct biological processes, e.g., TGFβ signalling pathway, providing new potential insights into the mechanisms of MTC metastasis.

Evaluation of breed-dependent differences in the innate immune responses of Holstein and Jersey cows toStaphylococcus aureusintramammary infection

Mastitis is one of the most prevalent diseases of cattle. Various studies have reported breed-dependent differences in the risk for developing this disease. Among two major breeds, Jersey cows have been identified as having a lower prevalence of mastitis than Holstein cows. It is well established that the nature of the initial innate immune response to infection influences the ability of the host to clear harmful bacterial pathogens. Whether differences in the innate immune response to intramammary infections explain, in part, the differential prevalence of mastitis in Holstein and Jersey cows remains unknown. The objective of the current study was to evaluate several parameters of the innate immune response of Holstein and Jersey cows to intramammary infection withStaphylococcus aureus, a common mastitis-inducing pathogen. To control for non-breed related factors that could influence these parameters, all cows were of the same parity, in similar stages of milk production, housed and managed under identical conditions, and experimentally infected and sampled in parallel. The following parameters of the innate immune response were evaluated: acute phase protein synthesis of serum amyloid A and lipopolysaccharide-binding protein; total and differential circulating white blood cell counts; milk somatic cell counts; mammary vascular permeability; milk N-acetyl-beta-d-glucosaminidase (NAGase) activity; and production of the cytokines, interferon (IFN)-γ, interleukin (IL)-12, tumour growth factor(TGF)-α, and TGF-β1. The temporal response of all of these parameters following infection was similar between Holstein and Jersey cows. Further, with the exception of changes in circulating neutrophils and NAGase activity, the overall magnitude of these parameters were also comparable. Together, these data demonstrate that the innate immune response of Holstein and Jersey cows toStaph. aureusintramammary infection remains highly conserved despite previously reported differences in mastitis prevalence, as well as genotypic and phenotypic traits, that exist between the two breeds.

CD8 + T cells reduce in vitro interferon-g production in Theiler’s murine encephalomyelitis virus-induced demyelinating disease model

In the Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease model for multiple sclerosis, regulatory CD8- T cells prevent demyelinating disease and reducein vivo interferon (IFN)-g production by anti-TMEV CD4- blast cells in BALB/c mice. We describe here that regulatory CD8- T cells reduce in vitro IFN-g production by lymph node cells from both TMEV and fowl gamma globulin immunized mice without affecting interleukin (IL)-4, IL-10, tumour growth factor-b or tumour necrosis factor-a production.