Role of the Fractalkine Receptor in CNS Autoimmune Inflammation: New Approach Utilizing a Mouse Model Expressing the Human CX3CR1

Multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS) is the leading cause of non-traumatic neurological disability in young adults. Immune mediated destruction of myelin and oligodendrocytes is considered the primary pathology of MS, but progressive axonal loss is the major cause of neurological disability. In an effort to understand microglia function during CNS inflammation, our laboratory focuses on the fractalkine/CX3CR1 signaling as a regulator of microglia neurotoxicity in various models of neurodegeneration. Fractalkine (FKN) is a transmembrane chemokine expressed in the CNS by neurons and signals through its unique receptor CX3CR1 present in microglia. During experimental autoimmune encephalomyelitis (EAE), CX3CR1 deficiency confers exacerbated disease defined by severe inflammation and neuronal loss. The CX3CR1 human polymorphism I249/M280 present in ∼20% of the population exhibits reduced adhesion for FKN conferring defective signaling whose role in microglia function and influence on neurons during MS remains unsolved. The aim of this study is to assess the effect of weaker signaling through hCX3CR1I249/M280 during EAE. We hypothesize that dysregulated microglial responses due to impaired CX3CR1 signaling enhance neuronal/axonal damage. We generated an animal model replacing the mouse CX3CR1 locus for the hCX3CR1I249/M280 variant. Upon EAE induction, these mice exhibited exacerbated EAE correlating with severe inflammation and neuronal loss. We also observed that mice with aberrant CX3CR1 signaling are unable to produce FKN and ciliary neurotrophic factor during EAE in contrast to wild type mice. Our results provide validation of defective function of the hCX3CR1I249/M280 variant and the foundation to broaden the understanding of microglia dysfunction during neuroinflammation. © 2018 Cardona et al

Presence of extracellular DNA in candida albicans biofilm matrix and its role in biofilm structure and antifungal susceptibility

Biofilms are structurally complex microconsortia of surface adhering cells embedded within an extracellular matrix (ECM) composed of substances produced and secreted by cells or derived from cell lysis. One of the recently discovered bacterial biofilms ECM components is the extracellular DNA (eDNA). Although the investigation on eDNA in fungal biofilms is scarce, preliminary studies suggest that eDNA may play a role in biofilms formed by the opportunistic fungal pathogen Candida albicans. Thus, the present study aimed at determining the eDNA content of C. albicans SC5314 biofilm ECM and the effect of DNase I treatment on biofilm formation and biofilm cells susceptibility to antifungals, as indicators of the role of eDNA in C. albicans biofilms. Results from our experiments showed that the ECM of C. albicans biofilms formed under conditions of flow for 48 h contained 3045.4 ± 227.3 ng eDNA/mg of protein. Additionally, using a microtiter plate model, we observed that different DNase treatments (0.02 - 2 mg/ml) did not affect further biofilm development by C. albicans adherent cells. However, DNase (> 0.03 mg/ml) promoted a general biomass reduction on C. albicans preformed biofilms. Finally, DNase (0.13 mg/ml) did not change C. albicans biofilm cells susceptibility to fluconazole, but increased their susceptibility to amphotericin B and caspofungin, as indicated by the lower SMIC compared to biofilms grown without DNase. This work presents evidence for the role of eDNA in C. albicans biofilm integrity and antifungal resistance consistent with eDNA being a key element of the ECM

Game and player: C. albicans biofilm lifestyle and extracellular DNA

DNA is as a structural component of bacterial biofilms extracellular matrix (ECM). Although evidences have shown that DNA may play a role in C. albicans biofilms, further studies are required to understand the contribution of extracellular DNA (eDNA) in C. albicans biofilm lifestyle. Herein we aimed to determine the eDNA content of C. albicans SC5314 biofilm ECM and the effect of DNase I and exogenous DNA treatments on biofilm formation and biofilm cells susceptibility to antifungals. First, for eDNA estimation in C. albicans biofilm ECM, biofilms were formed under flow conditions for 48 h. ECM was isolated and its DNA and protein contents were determined. Second, DNase (0.02 - 2 mg/ml) and exogenous DNA (10 - 2560 ng/ml) were added at different stages of biofilm development (microtiter plate model under static conditions). The effect of 24 h treatments was evaluated in terms of biofilm biomass by crystal violet assay (A550). Third, for antifungal testing, biofilms (in 96-well plates) were challenged with amphotericin B (0.06 - 16 mg/l), caspofungin (0.008 to 2 mg/l), and fluconazole (4 - 1024 mg/l) alone or in combination with DNase (0.125 mg/ml) or exogenous DNA (320 ng/ml). Sessile minimum inhibitory concentrations (SMIC) were determined at 80 % inhibition compared to drug-free controls using the XTT reduction assay. RPMI medium was used in all the assays. On one hand, C. albicans biofilms ECM contained 3045.4 ± 227.3 ng eDNA/mg of protein. On the other hand, DNase or exogenous DNA treatments did not affect further biofilm development by C. albicans adherent cells. In contrast, DNase (> 0.03 mg/ml) promoted a general biomass reduction on C. albicans preformed biofilms, as indicated by the reduction of A550 compared with the control. Furthermore addition of exogenous DNA (> 160 ng/ml) to preformed biofilms led to an increase in biofilm biomass, similarly assessed by the higher A550 readings compared with control biofilms. Finally, DNase I (0.125 mg/ml) did not change C. albicans biofilm cells susceptibility to fluconazole, but increased their susceptibility to amphotericin B and caspofungin, as indicated by the lower SMIC compared to biofilms grown without DNase. In contrast, exogenous DNA (320 ng/ml) did not affect C. albicans biofilm cells susceptibility against these antifungals

Albuminuria Changes and Cardiovascular and Renal Outcomes in Type 1 Diabetes: The DCCT/EDIC Study.

Background and objectives In trials of people with type 2 diabetes, albuminuria reduction with renin-angiotensin system inhibitors is associated with lower risks of cardiovascular events and CKD progression. We tested whether progression or remission of microalbuminuria is associated with cardiovascular and renal risk in a well characterized cohort of type 1 diabetes. Design, setting, participants, & measurements We studied 1441 participants in the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications study. Albumin excretion rate (AER) was quantified annually or biennially for up to 30 years. For each participant, albuminuria status was defined over time as normoalbuminuria (AER continuously \u3c30 mg/d), sustained microalbuminuria (AER, 30–299 mg/d on two consecutive visits), macroalbuminuria (AER≥300 mg/d), or remitted microalbuminuria (transition from sustained microalbuminuria to AER\u3c30 mg/d on two consecutive visits). We tested associations of time-updated albuminuria status with adjudicated clinical cardiovascular events, the development of reduced GFR (\u3c60 ml/min per 1.73 m2 on two consecutive visits), and subclinical cardiovascular disease. Results At least one cardiovascular event occurred in 184 participants, and 98 participants developed reduced eGFR. Compared with normoalbuminuria, sustained microalbuminuria, remitted microalbuminuria, and macroalbuminuria were each associated with higher risk of cardiovascular events (adjusted hazard ratios [HRs] and 95% confidence intervals [95% CIs]: 1.79 [1.13 to 2.85], 2.62 [1.68 to 4.07], and 2.65 [1.68 to 4.19], respectively) and reduced eGFR (adjusted HRs [95% CIs], 5.26 [2.43 to 11.41], 4.36 [1.80 to 10.57], and 54.35 [30.79 to 95.94], respectively). Compared with sustained microalbuminuria, remission to normoalbuminuria was not associated with reduced risk of cardiovascular events (adjusted HR, 1.33; 95% CI, 0.68 to 2.59) or reduced eGFR (adjusted HR, 1.75; 95% CI, 0.56 to 5.49). Compared with normoalbuminuria, sustained microalbuminuria, remitted microalbuminuria, and macroalbuminuria were associated with greater carotid intima-media thickness, and macroalbuminuria was associated with a greater degree of coronary artery calcification. Conclusions In type 1 diabetes, microalbuminuria and macroalbuminuria are associated with higher risks of cardiovascular disease and reduced eGFR, but achieving a remission of established microalbuminuria to normoalbuminuria does not appear to improve outcomes

Presence of extracellular DNA in the Candida albicans biofilm matrix and its contribution to biofilms

DNA has been described as a structural component of the extracellular matrix (ECM) in bacterial biofilms. In Candida albicans, there is a scarce knowledge concerning the contribution of extracellular DNA (eDNA) to biofilm matrix and overall structure. This work examined the presence and quantified the amount of eDNA in C. albicans biofilm ECM and the effect of DNase treatment and the addition of exogenous DNA on C. albicans biofilm development as indicators of a role for eDNA in biofilm development. We were able to detect the accumulation of eDNA in biofilm ECM extracted from C. albicans biofilms formed under conditions of flow, although the quantity of eDNA detected differed according to growth conditions, in particular with regards to the medium used to grow the biofilms. Experiments with C. albicans biofilms formed statically using a microtiter plate model indicated that the addition of exogenous DNA (>160 ng/ml) increases biofilm biomass and, conversely, DNase treatment (>0.03 mg/ml) decreases biofilm biomass at later time points of biofilm development. We present evidence for the role of eDNA in C. albicans biofilm structure and formation, consistent with eDNA being a key element of the ECM in mature C. albicans biofilms and playing a predominant role in biofilm structural integrity and maintenance.National Institute of Dental & Craniofacial ResearchFundação para a Ciência e Tecnologia (FCT) - SFRH/BD/28222/2006National Institute of Allergy and Infectious Disease

Systematic In Vivo Analysis of the Intrinsic Determinants of Amyloid β Pathogenicity

Protein aggregation into amyloid fibrils and protofibrillar aggregates is associated with a number of the most common neurodegenerative diseases. We have established, using a computational approach, that knowledge of the primary sequences of proteins is sufficient to predict their in vitro aggregation propensities. Here we demonstrate, using rational mutagenesis of the Aβ42 peptide based on such computational predictions of aggregation propensity, the existence of a strong correlation between the propensity of Aβ42 to form protofibrils and its effect on neuronal dysfunction and degeneration in a Drosophila model of Alzheimer disease. Our findings provide a quantitative description of the molecular basis for the pathogenicity of Aβ and link directly and systematically the intrinsic properties of biomolecules, predicted in silico and confirmed in vitro, to pathogenic events taking place in a living organism

Role of the Fractalkine Receptor in CNS Autoimmune Inflammation: New Approach Utilizing a Mouse Model Expressing the Human CX3CR1I249/M280 Variant

Multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS) is the leading cause of non-traumatic neurological disability in young adults. Immune mediated destruction of myelin and oligodendrocytes is considered the primary pathology of MS, but progressive axonal loss is the major cause of neurological disability. In an effort to understand microglia function during CNS inflammation, our laboratory focuses on the fractalkine/CX3CR1 signaling as a regulator of microglia neurotoxicity in various models of neurodegeneration. Fractalkine (FKN) is a transmembrane chemokine expressed in the CNS by neurons and signals through its unique receptor CX3CR1 present in microglia. During experimental autoimmune encephalomyelitis (EAE), CX3CR1 deficiency confers exacerbated disease defined by severe inflammation and neuronal loss. The CX3CR1 human polymorphism I249/M280 present in ∼20% of the population exhibits reduced adhesion for FKN conferring defective signaling whose role in microglia function and influence on neurons during MS remains unsolved. The aim of this study is to assess the effect of weaker signaling through hCX3CR1I249/M280 during EAE. We hypothesize that dysregulated microglial responses due to impaired CX3CR1 signaling enhance neuronal/axonal damage. We generated an animal model replacing the mouse CX3CR1 locus for the hCX3CR1I249/M280 variant. Upon EAE induction, these mice exhibited exacerbated EAE correlating with severe inflammation and neuronal loss. We also observed that mice with aberrant CX3CR1 signaling are unable to produce FKN and ciliary neurotrophic factor during EAE in contrast to wild type mice. Our results provide validation of defective function of the hCX3CR1I249/M280 variant and the foundation to broaden the understanding of microglia dysfunction during neuroinflammation

A Limited Role for Suppression in the Central Field of Individuals with Strabismic Amblyopia.

yesBackground: Although their eyes are pointing in different directions, people with long-standing strabismic amblyopia typically do not experience double-vision or indeed any visual symptoms arising from their condition. It is generally believed that the phenomenon of suppression plays a major role in dealing with the consequences of amblyopia and strabismus, by preventing images from the weaker/deviating eye from reaching conscious awareness. Suppression is thus a highly sophisticated coping mechanism. Although suppression has been studied for over 100 years the literature is equivocal in relation to the extent of the retina that is suppressed, though the method used to investigate suppression is crucial to the outcome. There is growing evidence that some measurement methods lead to artefactual claims that suppression exists when it does not. Methodology/Results: Here we present the results of an experiment conducted with a new method to examine the prevalence, depth and extent of suppression in ten individuals with strabismic amblyopia. Seven subjects (70%) showed no evidence whatsoever for suppression and in the three individuals who did (30%), the depth and extent of suppression was small. Conclusions: Suppression may play a much smaller role in dealing with the negative consequences of strabismic amblyopia than previously thought. Whereas recent claims of this nature have been made only in those with micro-strabismus our results show extremely limited evidence for suppression across the central visual field in strabismic amblyopes more generally. Instead of suppressing the image from the weaker/deviating eye, we suggest the visual system of individuals with strabismic amblyopia may act to maximise the possibilities for binocular co-operation. This is consistent with recent evidence from strabismic and amblyopic individuals that their binocular mechanisms are intact, and that, just as in visual normals, performance with two eyes is better than with the better eye alone in these individuals