Immunohistochemical identification of MMP-2 and MMP-9 in human dentin: correlative FEI-SEM/TEM analysis

Matrix metalloproteinases (MMPs) are a family of peptidases trapped within mineralized dentin matrix and involved with degradation of the extracellular matrix components in hybrid layers and caries. Despite their identification through indirect evidences and biochemical assays, MMP-2 and -9 have not been localized within the human dentin extracellular organic matrix. Thus, this study aimed to assess the localization and distribution of MMP-2 and -9 in human dentin organic matrix by employing a correlative field emission in-lens-scanning electron microscopy (FEI-SEM) and transmission electron microscopy (TEM) immunohistochemical approach. Dentin specimens were submitted either to a preembedding or to a postembedding immunolabeling technique using primary monoclonal antibodies anti-MMP-2 and anti-MMP-9 and exposed to a secondary antibody conjugated with gold nanoparticles. MMP-2 and -9 labelings were identified in the demineralized dentin matrix as highly electron-dense gold particles dispersed on the collagen fibrils. Correlative FEI-SEM/TEM observations confirmed that MMP-2 and MMP-9 are endogenous components of the human dentin organic matrix and revealed the three-dimensional relationship between these proteinases and the collagen fibrils, showing that both antibodies yielded a similar labeling pattern. In conclusion, the results of the study contribute to reveal distinct distribution pattern of gelatinases and support the hypothesis that these enzymes are intrinsic constituents of the dentin organic matrix after decalcification.     

Membrane permeability properties of dental adhesive films

This study evaluated the permeability properties of five experimental resin membranes that ranged from relatively hydrophobic to relatively hydrophilic to seal acid-etched dentin saturated with water or ethanol. The experimental resins (R1, R2, R3, R4, and R5) were evaluated as neat bonding agents or as solutions solvated with ethanol (70% resin/30% ethanol). The quality of dentin sealing by these experimental resins was expressed in terms of reflection coefficients calculated as the ratio of the effective osmotic pressure to the theoretical osmotic pressure of test solutions. The effective osmotic pressure produced across resin-bonded dentin was induced in hypertonic solutions (CaCl(2) or albumin) at zero hydrostatic pressure. The outward fluid flow induced by these solutions was brought to zero by applying an opposing negative hydrostatic pressure. The least hydrophilic resins blends, R1 and R2, exhibited significantly (p < 0.05) higher reflection coefficients than the most hydrophilic resins (R4 and R5) in both conditions of dentin saturation (water and ethanol). The reflection coefficients of neat resins were, in general, significantly higher when compared with their corresponding solvated versions in both conditions of dentin saturation. In dentin saturated with ethanol, bonding with neat or solvated resins, resulted in reflection coefficients that were significantly higher when compared with the results obtained in dentin saturated with water. Reflection coefficients of CaCl(2) (ca. 1 x 10(-4)) were significantly lower (p < 0.05) than for albumin (ca. 3 x 10(-2)). Application of hydrophobic resins may provide better sealing of acid-etched dentin if the substrate is saturated with ethanol, instead of water.     

Rho GTPase-mediated pathways in mature CD4+ T cells

Effective immune responses require the appropriate activation and differentiation of peripheral CD4(+) T cells. These processes need to be followed by the timely elimination of the responding T cells in order to restore T cell homeostasis. Defects in the appropriate regulation of T cell activation, expansion, and survival underlie the pathogenesis of many autoimmune disorders including SLE. The molecular machinery employed by T cells to properly control these processes and prevent the onset of autoimmunity has not been fully elucidated. Rho GTPases (which include the Rac, Cdc42, and Rho subfamilies) are molecular switches that control a wide range of cellular processes. Their fundamental role in biology is due to their ability to regulate both cytoskeletal dynamics and a large number of signal transduction pathways. Activation of Rho GTPases is now recognized as a key event in the coordination of immune responses and, particularly, in the activation of T cells. In this review, we will first provide an overview of the role of Rho GTPase-mediated pathways in mature CD4(+) T cells and then we will discuss recent studies, which suggest that deregulation of these pathways may play a role in the pathogenesis of SLE.     

Clinical, laboratory, diagnostic and prognostic aspects of canine lymphoma: a retrospective study

During the period of 8 years, 120 dogs affected by lymphoma were referred to the veterinary teaching hospital of the University. Canine lymphoma was classified and staged using a standardised diagnostic approach that involved the acquisition of detailed clinical history, physical examination and extended laboratory workup including lymph node cytology. Additionally, immunophenotyping was available in 22 cases. Multicentric lymphoma was the most common form of disease identified clinically, while the cytological classification revealed the B-cell polymorphic centroblastic subtype as the most frequent type of lymphoma in the study. Furthermore, the influence of several prognostic factors in relation to remission rate, relapsing rate and survival time were evaluated for 16 patients treated with a specific chemotherapy protocol and for which a complete follow-up was available. The results of our study suggest that chronic inflammation may represent a negative prognostic factor shortening both the relapsing and the survival times, while pre-treatment with steroids may have a negative effect on the survival time.     

Accumulation of neutral lipids in peripheral blood mononuclear cells as a distinctive trait of Alzheimer patients and asymptomatic subjects at risk of disease

Varicella is a common viral disease affecting almost the entire birth cohort. Although usually self-limiting, some cases of varicella can be serious, with 2 to 6% of cases attending a general practice resulting in complications. The hospitalisation rate for varicella in Europe ranges from 1.3 to 4.5 per 100,000 population/year and up to 10.1% of hospitalised patients report permanent or possible permanent sequelae (for example, scarring or ataxia). However, in many countries the epidemiology of varicella remains largely unknown or incomplete. In countries where routine childhood vaccination against varicella has been implemented, it has had a positive effect on disease prevention and control. Furthermore, mathematical models indicate that this intervention strategy may provide economic benefits for the individual and society. Despite this evidence and recommendations for varicella vaccination by official bodies such as the World Health Organization, and scientific experts in the field, the majority of European countries (with the exception of Germany and Greece) have delayed decisions on implementation of routine childhood varicella vaccination, choosing instead to vaccinate high-risk groups or not to vaccinate at all. In this paper, members of the Working Against Varicella in Europe group consider the practicalities of introducing routine childhood varicella vaccination in Europe, discussing the benefits and challenges of different vaccination options (vaccination vs. no vaccination, routine vaccination of infants vs. vaccination of susceptible adolescents or adults, two doses vs. one dose of varicella vaccine, monovalent varicella vaccines vs. tetravalent measles, mumps, rubella and varicella vaccines, as well as the optimal interval between two doses of measles, mumps, rubella and varicella vaccines). Assessment of the epidemiology of varicella in Europe and evidence for the effectiveness of varicella vaccination provides support for routine childhood programmes in Europe. Although European countries are faced with challenges or uncertainties that may have delayed implementation of a childhood vaccination programme, many of these concerns remain hypothetical and with new opportunities offered by combined measles, mumps, rubella and varicella vaccines, reassessment may be timely.     

Farnesoid X Receptor Critically Determines the Fibrotic Response in Mice but Is Expressed to a Low Extent in Human Hepatic Stellate Cells and Periductal Myofibroblasts

The nuclear bile acid receptor, farnesoid X receptor (FXR), may play a pivotal role in liver fibrosis. We tested the impact of genetic FXR ablation in four different mouse models. Hepatic fibrosis was induced in wild-type and FXR knock-out mice (FXR^−/−) by CCl₄ intoxication, 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding, common bile duct ligation, or Schistosoma mansoni (S.m.)-infection. In addition, we determined nuclear receptor expression levels (FXR, pregnane X receptor (PXR), vitamin D receptor, constitutive androstane receptor (CAR), small heterodimer partner (SHP)) in mouse hepatic stellate cells (HSCs), portal myofibroblasts (MFBs), and human HSCs. Cell type-specific FXR protein expression was determined by immunohistochemistry in five mouse models and prototypic human fibrotic liver diseases. Expression of nuclear receptors was much lower in mouse and human HSCs/MFBs compared with total liver expression with the exception of vitamin D receptor. FXR protein was undetectable in mouse and human HSCs and MFBs. FXR loss had no effect in CCl₄-intoxicated and S.m.-infected mice, but significantly decreased liver fibrosis of the biliary type (common bile duct ligation, 3,5-diethoxycarbonyl-1,4-dihydrocollidine). These data suggest that FXR loss significantly reduces fibrosis of the biliary type, but has no impact on non-cholestatic liver fibrosis. Since there is no FXR expression in HSCs and MFBs in liver fibrosis, our data indicate that these cells may not represent direct therapeutic targets for FXR ligands.The farnesoid X receptor (FXR;NR1H4) is a key regulator of hepatic bile acid homeostasis, lipoprotein and glucose metabolism, bacterial colonization of the small intestine, the inflammatory response, and liver regeneration.^1,2,3 Hereditary and acquired FXR defects may contribute to cholestasis and gallstone formation in humans.^4,5,6,7 Defects in its target genes (eg, bile salt export pump/ABCB11; multidrug resistance gene 3/ABCB4 (a phosphatidylcholine floppase); multidrug related protein 2/ABCC2) cause well-characterized clinical syndromes.^8,9,10,11 Moreover, FXR knockout mice (FXR^−/−) have impaired resistance to bile acid feeding,^12,13 and show substantial differences in the cholestatic phenotype in response to common bile duct ligation,^14,15,16 have increased susceptibility for diet-induced gallstone disease,^17,18 and impaired liver regeneration following partial hepatectomy.¹⁹ FXR may also directly or indirectly (eg, by the interaction with other members of the nuclear receptor family such as PXR/NR1I2 and VDR/NR1I1) regulate the metabolism and hepatic clearance of xenobiotics.^20,21,22Recent studies also reported mRNA expression of FXR in hepatic stellate cells and FXR protein in renal proximal tubules^23,24,25 suggesting that FXR could represent a therapeutic target for the treatment of liver fibrosis and diabetic nephropathy.^23,24,25,26 Moreover, FXR ligands were claimed to repress collagen expression in HSCs in vitro via a postulated FXR/SHP-dependent mechanism.²³ It is also attractive to hypothesis that genetic FXR variants may predispose patients suffering from various forms of liver diseases to liver fibrosis as a kind of genetic disease modifier.^7,27 Taken together its pleiotrophic functions (eg, central regulator of bile acid homoeostasis, glucose and lipid metabolism, inflammation) make FXR an extremely attractive candidate for therapeutic targeting in cholestatic liver diseases and nonalcoholic fatty liver disease including their major sequel liver fibrosis.^28,30 However, little is known on hepatic cell-type FXR expression in human liver fibrosis.The aims of this study were threefold. First, we aimed to determine the impact of genetic FXR ablation on the degree of liver fibrosis in untreated mice and four different well established mouse models including CCl₄-intoxicated mice, 3,5 -diethoxycarbonyl-1,4-dihydrocollidine (DDC)-intoxicated mice and common bile duct-ligated (CBDL) mice for biliary fibrosis, and infection with Schistosoma mansoni (S.m.), which has been shown to induce “pipe-stem” fibrosis and granuloma formation.^31,32 Comparison of cholestatic (DDC, CBDL) and non-cholestatic (CCl₄, S.m.) mouse models for liver fibrosis should provide differentiated knowledge on the role of FXR in various types and etiologies of liver fibrosis. Based on previous studies reporting that pharmacological activation of FXR is antifibrotic in liver but also kidney^23,25 we hypothesized that FXR^−/− mice spontaneously develop liver fibrosis and are more susceptible to experimentally induced liver fibrosis due to the lack of a postulated FXR/SHP-dependent down-regulation of collagen mRNA expression in profibrotic states.^23,24 We therefore compared the extent of fibrosis in FXR^−/− mice and wild-type controls in a longitudinal study under baseline conditions and in response to cholestatic and non-cholestatic fibrogenic injury. Second, we aimed to determine the expression of genes involved in bile acid transport/metabolism and their regulatory nuclear receptors (including FXR, PXR, CAR/NR1I3, VDR, and SHP/NR0B2) in isolated profibrogenic rodent cells [ie, periductal myofibroblasts (MFBs), and quiescent as well as activated hepatic stellate cells (HSCs)] and to test the effects of FXR ligands on FXR target genes in vitro. Cell type-specific FXR protein expression was determined in five different in vivo models for liver fibrosis. Finally, we cross-validated these findings in isolated human HSCs and histological sections from human prototypic fibrotic liver diseases [eg, primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), and alcoholic steatohepatitis (ASH)].     

The patterns of spontaneous Ca2+ signals generated by ventral spinal neurons in vitro show time-dependent refinement

Embryonic spinal neurons maintained in organotypic slice culture are known to mimic certain maturation-dependent signalling changes. With such a model we investigated, in embryonic mouse spinal segments, the age-dependent spatio-temporal control of intracellular Ca²⁺ signalling generated by neuronal populations in ventral circuits and its relation with electrical activity. We used Ca²⁺ imaging to monitor areas located within the ventral spinal horn at 1 and 2 weeks of in vitro growth. Primitive patterns of spontaneous neuronal Ca²⁺ transients (detected at 1 week) were typically synchronous. Remarkably, such transients originated from widespread propagating waves that became organized into large-scale rhythmic bursts. These activities were associated with the generation of synaptically mediated inward currents under whole-cell patch-clamp. Such patterns disappeared during longer culture of spinal segments: at 2 weeks in culture, only a subset of ventral neurons displayed spontaneous, asynchronous and repetitive Ca²⁺ oscillations dissociated from background synaptic activity. We observed that the emergence of oscillations was a restricted phenomenon arising together with the transformation of ventral network electrophysiological bursting into asynchronous synaptic discharges. This change was accompanied by the appearance of discrete calbindin immunoreactivity against an unchanged background of calretinin-positive cells. It is attractive to assume that periodic oscillations of Ca²⁺ confer a summative ability to these cells to shape the plasticity of local circuits through different changes (phasic or tonic) in intracellular Ca²⁺.     

Exercise attenuates the clinical, synaptic and dendritic abnormalities of experimental autoimmune encephalomyelitis

Voluntary exercise is beneficial in models of primarily neurodegenerative disorders. Whether exercise also affects inflammatory neurodegeneration is unknown. In the present study, we evaluated the clinical, synaptic and neuropathological effects of voluntary wheel running in mice with myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. Exercising EAE mice exhibited less severe neurological deficits compared to control EAE animals. The sensitivity of striatal GABA synapses to the stimulation of cannabinoid CB1 receptors was dramatically downregulated following EAE induction, and was rescued by exercise in EAE mice with access to a running wheel. Finally, we found that exercise was able to contrast dendritic spine loss induced by EAE in striatal neurons, although the degree of inflammatory response was similar in the two experimental groups.Our work suggests that life style and experiences can impact the clinical course of inflammatory neurodegenerative diseases by affecting their synaptic bases.