Efficient Recruitment of Lymphocytes in Inflamed Brain Venules Requires Expression of Cutaneous Lymphocyte Antigen and Fucosyltransferase-VII

Abstract Lymphocyte migration into the brain represents a critical event in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the mechanisms controlling the recruitment of lymphocytes to the CNS via inflamed brain venules are poorly understood, and therapeutic approaches to inhibit this process are consequently few. In this study, we demonstrate for the first time that human and murine Th1 lymphocytes preferentially adhere to murine inflamed brain venules in an experimental model that mimics early inflammation during EAE. A virtually complete inhibition of rolling and arrest of Th1 cells in inflamed brain venules was observed with a blocking anti-P-selectin glycoprotein ligand 1 Ab and anti-E- and P-selectin Abs. Th1 lymphocytes produced from fucosyltransferase (FucT)-IV−/− mice efficiently tethered and rolled, whereas in contrast, primary adhesion of Th1 lymphocytes obtained from FucT-VII−/− or Fuc-VII−/−FucT-IV−/− mice was drastically reduced, indicating that FucT-VII is critical for the recruitment of Th1 cells in inflamed brain microcirculation. Importantly, we show that Abs directed against cutaneous lymphocyte Ag (CLA), a FucT-VII-dependent carbohydrate modification of P-selectin glycoprotein ligand 1, blocked rolling of Th1 cells. By exploiting a system that allowed us to obtain Th1 and Th2 cells with skin- vs gut-homing (CLA+ vs integrin β7+) phenotypes, we observed that induced expression of CLA on Th cells determined a striking increase of rolling efficiency in inflamed brain venules. These observations allow us to conclude that efficient recruitment of activated lymphocytes to the brain in the contexts mimicking EAE is controlled by FucT-VII and its cognate cell surface Ag CLA

Common and rare variants in TMEM175 gene concur to the pathogenesis of Parkinson’s Disease in Italian patients

Parkinson’s disease (PD) represents the most common neurodegenerative movement disorder. We recently identified 16 novel genes associated with PD. In this study, we focused the attention on the common and rare variants identified in the lysosomal K+ channel TMEM175. The study includes a detailed clinical and genetic analysis of 400 cases and 300 controls. Molecular studies were performed on patient-derived fibroblasts. The functional properties of the mutant channels were assessed by patch-clamp technique and co-immunoprecipitation. We have found that TMEM175 was highly expressed in dopaminergic neurons of the substantia nigra pars compacta and in microglia of the cerebral cortex of the human brain. Four common variants were associated with PD, including two novel variants rs2290402 (c.-10C > T) and rs80114247 (c.T1022C, p.M341T), located in the Kozak consensus sequence and TM3II domain, respectively. We also disclosed 13 novel highly penetrant detrimental mutations in the TMEM175 gene associated with PD. At least nine of these mutations (p.R35C, p. R183X, p.A270T, p.P308L, p.S348L, p. L405V, p.R414W, p.P427fs, p.R481W) may be sufficient to cause the disease, and the presence of mutations of other genes correlated with an earlier disease onset. In vitro functional analysis of the ion channel encoded by the mutated TMEM175 gene revealed a loss of the K+ conductance and a reduced channel affinity for Akt. Moreover, we observed an impaired autophagic/lysosomal proteolytic flux and an increase expression of unfolded protein response markers in patient-derived fibroblasts. These data suggest that mutations in TMEM175 gene may contribute to the pathophysiology of PD

A role for leukocyte-endothelial adhesion mechanisms in epilepsy

The mechanisms involved in the pathogenesis of epilepsy, a chronic neurological disorder that affects approximately 1 percent of the world population, are not well understood1–3. Using a mouse model of epilepsy, we show that seizures induce elevated expression of vascular cell adhesion molecules and enhanced leukocyte rolling and arrest in brain vessels mediated by the leukocyte mucin P-selectin glycoprotein ligand-1 (PSGL-1) and leukocyte integrins α4β1 and αLβ2. Inhibition of leukocyte-vascular interactions either with blocking antibodies, or in mice genetically deficient in functional PSGL-1, dramatically reduced seizures. Treatment with blocking antibodies following acute seizures prevented the development of epilepsy. Neutrophil depletion also inhibited acute seizure induction and chronic spontaneous recurrent seizures. Blood-brain barrier (BBB) leakage, which is known to enhance neuronal excitability, was induced by acute seizure activity but was prevented by blockade of leukocyte-vascular adhesion, suggesting a pathogenetic link between leukocyte-vascular interactions, BBB damage and seizure generation. Consistent with potential leukocyte involvement in the human, leukocytes were more abundant in brains of epileptics than of controls. Our results suggest leukocyte-endothelial interaction as a potential target for the prevention and treatment of epilepsy

A role for leukocyte-endothelial adhesion mechanisms in epilepsy

Il presente progetto di ricerca per il Dottorato in Scienze Biomediche Traslazionali ha avuto come obiettivo uno studio sperimentale delle implicazioni del reclutamento leucocitario nella patogenesi e nella evoluzione dell\u2019epilessia. I meccanismi coinvolti nella patogenesi dell\u2019epilessia, una patologia neurologica cronica che colpisce circa l\u2019uno percento della popolazione mondiale, non sono ancora chiari1-3. Tuttavia studi recenti suggeriscono che l\u2019infiammazione potrebbe giocare un ruolo nella patogenesi di questa malattia. Il reclutamento leucocitario \ue8 una caratteristica ed un punto di intervento terapeutico fondamentale nell\u2019infiammazione tissutale, ma un ruolo per le interazioni tra leucociti ed endotelio nella patogenesi delle crisi epilettiche non \ue8 ancora stato esplorato. Nel presente progetto abbiamo utilizzato un modello murino di epilessia, indotto da pilocarpina, ed abbiamo dimostrato che le crisi inducono a livello dei vasi cerebrali elevata espressione di molecole di adesione vascolari, come vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) e selectine. Mediante la tecnica della microscopia intravitale sono state evidenziate aumentate interazioni (rotolamento ed arresto) in vivo da parte di granulociti e leucociti TH1 con l\u2019endotelio cerebrale dopo le crisi, ed \ue8 stato osservato che queste interazioni sono mediate dalla mucina leucocitaria P-selectin glycoprotein ligand-1 (PSGL-1) e dalle integrine leucocitarie \u3b14\u3b21 e \u3b1L\u3b22. Il trattamento dei topi con anticorpi bloccanti queste molecole di adesione, ha indotto una drammatica riduzione delle crisi acute e croniche indotte da pilocarpina. Anche la deficienza genetica di PSGL-1 funzionale o di \u3b11-3-fucosiltransferasi, enzimi coinvolti nella glicosilazione del PSGL-1, inibisce le crisi. Per confermare ulteriormente un ruolo dei leucociti nell\u2019induzione delle crisi, abbiamo depleto i granulociti murini prima della somministrazione di pilocarpina e abbiamo nuovamente osservato una consistente riduzione delle crisi acute e di quelle croniche spontanee ricorrenti. La rottura della barriera emato-encefalica (BBB), che, come dimostrato da diversi recenti lavori, \ue8 noto aumentare l\u2019eccitabilit\ue0 neuronale, \ue8 stata drasticamente ridotta negli animali trattati con anticorpi anti-integrina \u3b14 o nei topi geneticamente deficienti di PSGL-1 o di fucosiltransferasi. Questo suggerisce un legame patogenetico tra: le interazioni tra leucociti e i vasi cerebrali, il danno di barriera e l\u2019insorgenza delle crisi. Abbiamo infine ottenuto dati che suggeriscono che l\u2019epilessia pu\uf2 essere correlata nei topi con elevate conte di leucociti in preparati immunoistochimici di cervelli dopo crisi. Sono stati inoltre da noi analizzati anche campioni di cervelli umani di pazienti epilettici dove abbiamo confermato una maggiore abbondanza di leucociti nei cervelli epilettici rispetto ai controlli. I nostri risultati indicano le interazioni tra leucociti ed endotelio cerebrale come un target potenziale per la prevenzione ed il trattamento dell\u2019epilessia.The present project represents an experimental study on the leukocyte recruitment in the induction and evolution of epilepsy. The mechanisms involved in the pathogenesis of epilepsy, a chronic neurological disorder that affects approximately one percent of the general world population, are not well understood1-3, but recent data suggest that inflammation may play a role in the pathogenesis of this disease. Leukocyte recruitment is a hallmark of and a point of therapeutic intervention in tissue inflammation, but a role for leukocyte-endothelial interactions in seizure pathogenesis has not been explored. Using a mouse model of epilepsy, pilocarpine induced, we show that seizures induce elevated expression of vascular cell adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and selectins on brain vessels. Intravital microscopy revealed enhanced granulocyte and TH1 lymphocyte interactions (rolling and arrest) in vivo with brain microvasculature after seizures, and these leukocyte-vascular interactions were mediated by the leukocyte mucin P-selectin glycoprotein ligand-1 (PSGL-1) and leukocyte integrins \u3b14\u3b21 and \u3b1L\u3b22. Treatment of mice with blocking antibodies dramatically reduced acute and chronic seizures induced by pilocarpine. Genetic deficiency of PSGL-1 or of the \u3b11-3-fucosyltransferases, enzymes involved in the glycosylation of PSGL-1, also inhibited seizures. To further confirm a role for leucocytes in the induction of seizures we performed granulocytes depletion in mice before pilocarpine administration and we again observed a consistent reduction of acute and chronic spontaneous recurrent seizures. Blood-brain barrier (BBB) leakage, which is known to enhance neuronal excitability as shown in different recent articles, was induced by acute seizure activity, but it was drastically reduced in anti-\u3b14 treated or PSGL-1 or FucT deficient animals, suggesting a pathogenetic link between leukocyte-vascular interactions, BBB damage and seizure generation. Finally, we provide data suggesting that epilepsy may be correlated in mice with elevated brain leukocyte counts after seizures. Consistent with the potential leukocyte involvement in epilepsy in humans, leukocytes were more abundant in brains of individuals with epilepsy than in controls. Our results suggest leukocyte-endothelial interaction as a potential target for the prevention and treatment of epilepsy

LC-HRMS-Based Non-Targeted Metabolomics for the Assessment of Honey Adulteration with Sugar Syrups: A Preliminary Study

Honey is a natural product that is in great demand and has a relatively high price, thus making it one of the most common targets of economically motivated adulteration. Its adulteration can be obtained by adding cheaper honey or sugar syrups or by overfeeding honeybees with sugar syrups. Adulteration techniques are constantly evolving and advanced techniques and instruments are required for its detection. We used non-targeted metabolomics to underscore potential markers of honey adulteration with sugar syrups. The metabolomic profiles of unadulterated honeys and sugar beet, corn and wheat syrups were obtained using hydrophilic interaction liquid chromatography high-resolution mass spectrometry (LC-HRMS). The potential markers have been selected after data processing. Fortified honey (5%, 10% and 20%), honey obtained from overfeeding, and 58 commercial honeys were analyzed. One potential marker appeared with a specific signal for syrups and not for honey. This targeted analysis showed a linear trend in fortified honeys with a calculated limit of quantification around 5% of fortification

ROLE OF FUCOSYLTRANSFERASE-VII AND P-SELECTIN GLYCOPROTEIN LIGAND-1 IN THE MIGRATION OF CD4+CD25+ REGULATORY CELLS IN INFLAMED BRAIN

CD4+CD25+ regulatory T (Treg) cells participate in immunologic homeostasis by active suppression of inappropriate immune responses and are able to inhibit a variety of autoimmune and inflammatory diseases. Treg cells inhibit the activation of autoreactive T cells and suppress organ-specific autoimmunity. The mechanisms of Treg cells involved in the regulation of experimental autoimmune encephalomyelitis (EAE) are not well understood. Recent studies have shown a direct involvment of Treg cells in the natural resolution of EAE within the central nervous system (CNS) and a strong correlation between their migration pattern and their ability to control inflammatory responses. However, the molecular mechanisms controlling the migration of Tregs in inflamed brain are not known. P-selectin glycoprotein ligand-1 (PSGL-1) and alpha (1,3) fucosyltransferases (FucT), enzymes that catalyze the glycosylation of PSGL-1 and control its functionality, are molecules involved in the migration of leukocytes in sites of inflammation. The GOAL of this study was to determine the role of PSGL-1 in the migration of Treg cells in mice with EAE. METHODS: Active and transfer EAE were performed in WT/C57Bl/6, FucT-VII and PSGL-1 deficient (FucT-VII-/- and PSGL-1-/-) mice using MOG35-55 peptide. CD4+CD25+ cells were obtained by magnetic cell sorting. Flow cytometry and ImageStream technology were used to determine the expression and distribution of adhesion molecules and binding capacity to P-selectin and E-selectin chimeras. Migration properties of WT, FucT-VII-/- and PSGL-1-/- Tregs were determined with in vivo migration assays using 3H-glycerol-labeled Tregs. Intravital microscopy experiments were performed in order to determine the ability of WT, FucT-VII-/- and PSGL-1-/- Tregs to interact with inflamed brain endothelium. RESULTS: Encephalitogenic T cells produced from FucT-VII-/- and PSGL-1-/- mice transferred a significantly more severe disease that WT T cells. We observed no significant differences in the expression of adhesion molecules and IL-4 and IFN-\u3b3 production of autoreactive T cells from PSGL-1 and FucT-VII deficient mice. However, co-cultures with CD4+CD25+ Tregs and effector cells showed that deficiency of PSGL-1 and FucT-VII leads to a marked decrease of suppression capacity of Tregs. Interestingly, activated CD4+CD25+ Tregs have increased expression of functional PSGL-1 and a significantly higher suppressor activity in vitro when compared to na\uefve cells. In addition, activated Tregs display increased migration capacity in inflamed brain in mice with EAE. Both activated and na\uefve WT Treg cells preferentially migrated into the CNS in the pre-clinical phase of active EAE, than at disease onset. Treg cells from FucT-VII-/- and PSGL-1-/- mice present decreased migration ability to inflamed CNS when compared to WT Tregs. Moreover, intravital microscopy experiments showed a dramatic decrease of adhesive interactions in inflamed brain microcirculation in FucT-VII-/- and PSGL-1-/- Tregs. Finally, Tregs deficient of PSGL-1 and FucT-VII displayed a reduced capacity to suppress active EAE when compared to WT cells. CONCLUSION: Our data demonstrate that PSGL-1 and the fucosylation of its glycans by FucT-VII are involved in the suppression mediated by CD4+CD25+ Treg cells in MOG-induced EAE. Moreover, in addition to a role in cell-cell contact required for efficient suppression, our results suggest a key role of PSGL-1 and FucT-VII activity in the recruitment of CD4+CD25+ cells into the brain of mice with EAE

The Honey Bee: An Active Biosampler of Environmental Pollution and a Possible Warning Biomarker for Human Health

Member states of the European Union are required to ensure the initiation of monitoring programs to verify honey bee exposure to pesticides, where and as appropriate. Based on 620 samples of dead honey bees—42 of pollen, 183 of honey and 32 of vegetables—we highlighted the presence, as analyzed by liquid and gas chromatography coupled with tandem mass spectrometric detection, of many active substances, mainly tau-fluvalinate, piperonyl butoxide, chlorpyrifos and chlorpyrifos-methyl, permethrin and imidacloprid. Among the active substances found in analyzed matrices linked to honey bee killing incidents, 38 belong to hazard classes I and II, as methiocarb, methomyl, chlorpyrifos, cypermethrin and permethrin, thus representing a potential risk for human health. We have shown that, at different times between 2015 and 2020, during implementation of the Italian national guidelines for managing reports of bee colony mortality or depopulation associated with pesticide use, pesticide pollution events occurred that could raise concern for human health. Competent authorities could, as part of a One Health approach, exploit the information provided by existing reporting programs on honey bees and their products, in view of the close correlation to human health, animal health and ecosystem health

Regulation of conformer-specific activation of the integrin LFA-1 by achemokine-triggered Rho signaling module.

Regulation of the affinity of the \u3b22 integrin LFA-1 by chemokines is critical to lymphocyte trafficking, but the signaling mechanisms that control this process are not well understood. Here we investigated the signaling events controlling LFA-1 affinity triggering by chemokines in human primary T lymphocytes. We found that the small GTPase Rac1 mediated chemokine-induced LFA-1 affinity triggering and lymphocyte arrest in high endothelial venules. Unexpectedly, another Rho family member, Cdc42, negatively regulated LFA-1 activation. The Rho effectors PLD1 and PIP5KC were also critical to LFA-1 affinity modulation. Notably, PIP5KC was found to specifically control the transition of LFA-1 from an extended low-intermediate state to a high-affinity state, which correlated with lymphocyte arrest. Thus, chemokines control lymphocyte trafficking by triggering a Rho-dependent signaling cascade leading to conformer-specific modulation of LFA-1 affinity

( Standard methods for Apis mellifera beeswax research)

Due to its multifunctional and complex role in the honey bee colony functioning and health (construction material allowing food storage, brood rearing, thermoregulation, mediation in chemical and mechanical communication, substrate for pathogens, toxins and waste), Apis mellifera beeswax has been widely studied over the last five decades. This is supported by a comprehensive set of scientific reports covering different aspects of beeswax research. In this article, we present an overview of the methods for studying chemical, biological, constructional, and quality aspects of beeswax. We provide a detailed description of the methods for investigating wax scales, comb construction and growth pattern, cell properties, chemical composition of beeswax using different analytical tools, as well as the analytical procedures for provenancing beeswax and beeswax-derived compounds based on the hydrogen isotope ratio (IRMS). Along with classical physico-chemical and sensory analysis, we describe more precise and accurate methods for detection of adulterants in beeswax (GC-MS and FTIR-ATR). Moreover, we present methods for studying the influence of beeswax (comb foundation) adulteration on comb construction. Analytical protocols for determining the pesticide residues using different chromatographic and spectroscopic techniques are also described. As beeswax is an agent of high risk for the transmission of bee diseases, we present methods for detection of pathogens in beeswax. To ensure the reproducibility of experiments and results, we present best practice approaches and detailed protocols for all methods described, as well as their advantages and disadvantages.COST ActionEuropean Cooperation in Science and Technology (COST) [FA0803]; Ricola Foundation - Nature CultureWe would like to acknowledge the Ricola Foundation - Nature & Culture, and COST Action FA0803 for funding the COLOSS (Prevention of honey bee COlony LOSSes) network and making the BEEBOOK possible. We would also like to express our gratitude to Editor Vincent Dietemann for his kind support all the way, and for providing helpful and constructive suggestions