The Flavonoid Luteolin Inhibits Fcγ-Dependent Respiratory Burst in Granulocytes, but Not Skin Blistering in a New Model of Pemphigoid in Adult Mice

Bullous pemphigoid is an autoimmune blistering skin disease associated with autoantibodies against the dermal-epidermal junction. Passive transfer of antibodies against BP180/collagen (C) XVII, a major hemidesmosomal pemphigoid antigen, into neonatal mice results in dermal-epidermal separation upon applying gentle pressure to their skin, but not in spontaneous skin blistering. In addition, this neonatal mouse model precludes treatment and observation of diseased animals beyond 2–3 days. Therefore, in the present study we have developed a new disease model in mice reproducing the spontaneous blistering and the chronic course characteristic of the human condition. Adult mice were pre-immunized with rabbit IgG followed by injection of BP180/CXVII rabbit IgG. Mice pre-immunized against rabbit IgG and injected 6 times every second day with the BP180/CXVII-specific antibodies (n = 35) developed spontaneous sustained blistering of the skin, while mice pre-immunized and then treated with normal rabbit IgG (n = 5) did not. Blistering was associated with IgG and complement C3 deposits at the epidermal basement membrane and recruitment of inflammatory cells, and was partly dependent on Ly-6G-positive cells. We further used this new experimental model to investigate the therapeutic potential of luteolin, a plant flavonoid with potent anti-inflammatory and anti-oxidative properties and good safety profile, in experimental BP. Luteolin inhibited the Fcγ-dependent respiratory burst in immune complex-stimulated granulocytes and the autoantibody-induced dermal-epidermal separation in skin cryosections, but was not effective in suppressing the skin blistering in vivo. These studies establish a robust animal model that will be a useful tool for dissecting the mechanisms of blister formation and will facilitate the development of more effective therapeutic strategies for managing pemphigoid diseases

Experimental models for the autoimmune and inflammatory blistering disease, Bullous pemphigoid

Bullous pemphigoid (BP) is a subepidermal skin blistering disease characterized immunohistologically by dermal-epidermal junction (DEJ) separation, an inflammatory cell infiltrate in the upper dermis, and autoantibodies targeted toward the hemidesmosomal proteins BP230 and BP180. Development of an IgG passive transfer mouse model of BP that reproduces these key features of human BP has demonstrated that subepidermal blistering is initiated by anti-BP180 antibodies and mediated by complement activation, mast cell degranulation, neutrophil infiltration, and proteinase secretion. This model is not compatible with study of human pathogenic antibodies, as the human and murine antigenic epitopes are not cross-reactive. The development of two novel humanized mouse models for the first time has enabled study of disease mechanisms caused by BP autoantibodies, and presents an ideal in vivo system to test novel therapeutic strategies for disease management

Thymidine phosphorylase expression in normal, hyperplastic and neoplastic prostates: correlation with tumour associated macrophages, infiltrating lymphocytes, and angiogenesis

Thymidine phosphorylase is an angiogenic factor primarily expressed by cancer cells, stromal cells and tumour-associated macrophages in many human malignancies. These different types of thymidine phosphorylase-expressing cells, however, may have a distinct place in the angiogenic process, and this question was addressed in the present study. A series of 20 normal/hyperplastic prostate glands and 60 prostate carcinomas was investigated by immunohistochemistry, using specific antibodies for thymidine phosphorylase (P-GF.44C), tumour-associated macrophages (CD68), endothelium (CD31) and prostate specific antigen (ER-PR8). Thymidine phosphorylase expression by normal and hyperplastic epithelial or stromal cells occurred almost exclusively in the context of an intense lymphocytic infiltrate. High thymidine phosphorylase cancer cells and thymidine phosphorylase stromal cells expression was associated with high angiogenesis in prostate carcinomas, and this significant association was extended to include both tumour-associated macrophages and tumour-infiltrating lymphocytes. Thymidine phosphorylase expression and tumour-infiltrating lymphocytes were related inversely with prostate specific antigen reactivity. In conclusion, thymidine phosphorylase is a major angiogenic factor in prostate carcinomas and its up-regulation is likely to occur in the context of a host immune response

Glycoprotein Hyposialylation Gives Rise to a Nephrotic-Like Syndrome That Is Prevented by Sialic Acid Administration in GNE V572L Point-Mutant Mice

Mutations in the key enzyme of sialic acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/N-acetyl-mannosamine kinase, result in distal myopathy with rimmed vacuoles (DMRV)/hereditary inclusion body myopathy (HIBM) in humans. Sialic acid is an acidic monosaccharide that modifies non-reducing terminal carbohydrate chains on glycoproteins and glycolipids, and it plays an important role in cellular adhesions and interactions. In this study, we generated mice with a V572L point mutation in the GNE kinase domain. Unexpectedly, these mutant mice had no apparent myopathies or motor dysfunctions. However, they had a short lifespan and exhibited renal impairment with massive albuminuria. Histological analysis showed enlarged glomeruli with mesangial matrix deposition, leading to glomerulosclerosis and abnormal podocyte foot process morphologies in the kidneys. Glycan analysis using several lectins revealed glomerular epithelial cell hyposialylation, particularly the hyposialylation of podocalyxin, which is one of important molecules for the glomerular filtration barrier. Administering Neu5Ac to the mutant mice from embryonic stages significantly suppressed the albuminuria and renal pathology, and partially recovered the glomerular glycoprotein sialylation. These findings suggest that the nephrotic-like syndrome observed in these mutant mice resulted from impaired glomerular filtration due to the hyposialylation of podocyte glycoproteins, including podocalyxin. Furthermore, it was possible to prevent the nephrotic-like disease in these mice by beginning Neu5Ac treatment during gestation

Characteristics of the Alternative Phenotype of Microglia/Macrophages and its Modulation in Experimental Gliomas

Microglia (brain resident macrophages) accumulate in malignant gliomas and instead of initiating the anti-tumor response, they switch to a pro-invasive phenotype, support tumor growth, invasion, angiogenesis and immunosuppression by release of cytokines/chemokines and extracellular matrix proteases. Using immunofluorescence and flow cytometry, we demonstrate an early accumulation of activated microglia followed by accumulation of macrophages in experimental murine EGFP-GL261 gliomas. Those cells acquire the alternative phenotype, as evidenced by evaluation of the production of ten pro/anti-inflammatory cytokines and expression profiling of 28 genes in magnetically-sorted CD11b+ cells from tumor tissues. Furthermore, we show that infiltration of implanted gliomas by amoeboid, Iba1-positive cells can be reduced by a systematically injected cyclosporine A (CsA) two or eight days after cell inoculation. The up-regulated levels of IL-10 and GM-CSF, increased expression of genes characteristic for the alternative and pro-invasive phenotype (arg-1, mt1-mmp, cxcl14) in glioma-derived CD11b+ cells as well as enhanced angiogenesis and tumor growth were reduced in CsA-treated mice. Our findings define for the first time kinetics and biochemical characteristics of glioma-infiltrating microglia/macrophages. Inhibition of the alternative activation of tumor-infiltrating macrophages significantly reduced tumor growth. Thus, blockade of microglia/macrophage infiltration and their pro-invasive functions could be a novel therapeutic strategy in malignant gliomas

Evaluating the SERCA2 and VEGF mRNAs as Potential Molecular Biomarkers of the Onset and Progression in Huntington's Disease

Abnormalities of intracellular Ca2+ homeostasis and signalling as well as the down-regulation of neurotrophic factors in several areas of the central nervous system and in peripheral tissues are hallmarks of Huntington\u2019s disease (HD). As there is no therapy for this hereditary, neurodegenerative fatal disease, further effort should be made to slow the progression of neurodegeneration in patients through the definition of early therapeutic interventions. For this purpose, molecular biomarker(s) for monitoring disease onset and/or progression and response to treatment need to be identified. In the attempt to contribute to the research of peripheral candidate biomarkers in HD, we adopted a multiplex real-time PCR approach to analyse the mRNA level of targeted genes involved in the control of cellular calcium homeostasis and in neuroprotection. For this purpose we recruited a total of 110 subjects possessing the HD mutation at different clinical stages of the disease and 54 sex- and agematched controls. This study provides evidence of reduced transcript levels of sarco-endoplasmic reticulum-associated ATP2A2 calcium pump (SERCA2) and vascular endothelial growth factor (VEGF) in peripheral blood mononuclear cells (PBMCs) of manifest and premanifest HD subjects. Our results provide a potentially new candidate molecular biomarker for monitoring the progression of this disease and contribute to understanding some early events that might have a role in triggering cellular dysfunctions in HD

Glucosinolates, myrosinase hydrolysis products, and flavonols found in rocket (Eruca sativa and Diplotaxis tenuifolia)

Rocket species have been shown to have very high concentrations of glucosinolates and flavonols, which have numerous positive health benefits with regular consumption. In this review we highlight how breeders and processors of rocket species can utilize genomic and phytochemical research to improve varieties and enhance the nutritive benefits to consumers. Plant breeders are increasingly looking to new technologies such as HPLC, UPLC, LC-MS and GC-MS to screen populations for their phytochemical content to inform plant selections. Here we collate the research that has been conducted to-date in rocket, and summarise all glucosinolate and flavonol compounds identified in the species. We emphasize the importance of the broad screening of populations for phytochemicals and myrosinase degradation products, as well as unique traits that may be found in underutilized gene bank resources. We also stress that collaboration with industrial partners is becoming essential for long-term plant breeding goals through research

Astrocytes: biology and pathology

Astrocytes are specialized glial cells that outnumber neurons by over fivefold. They contiguously tile the entire central nervous system (CNS) and exert many essential complex functions in the healthy CNS. Astrocytes respond to all forms of CNS insults through a process referred to as reactive astrogliosis, which has become a pathological hallmark of CNS structural lesions. Substantial progress has been made recently in determining functions and mechanisms of reactive astrogliosis and in identifying roles of astrocytes in CNS disorders and pathologies. A vast molecular arsenal at the disposal of reactive astrocytes is being defined. Transgenic mouse models are dissecting specific aspects of reactive astrocytosis and glial scar formation in vivo. Astrocyte involvement in specific clinicopathological entities is being defined. It is now clear that reactive astrogliosis is not a simple all-or-none phenomenon but is a finely gradated continuum of changes that occur in context-dependent manners regulated by specific signaling events. These changes range from reversible alterations in gene expression and cell hypertrophy with preservation of cellular domains and tissue structure, to long-lasting scar formation with rearrangement of tissue structure. Increasing evidence points towards the potential of reactive astrogliosis to play either primary or contributing roles in CNS disorders via loss of normal astrocyte functions or gain of abnormal effects. This article reviews (1) astrocyte functions in healthy CNS, (2) mechanisms and functions of reactive astrogliosis and glial scar formation, and (3) ways in which reactive astrocytes may cause or contribute to specific CNS disorders and lesions