Antigen-presentation of non-peptidic antigens lipid trafficking and loading

T cells recognize a broad variety of antigens, including peptides, lipids and non-peptidic phosphorylated metabolites. Clarification of the rules rendering non-peptidic molecules immunogenic is essential to understand and to influence the reactions of the immune system to this class of substances in health and disease. Despite recent advances in research about immune responses to non-peptidic compounds, important issues remain unanswered. Molecular mechanisms governing the immunogenicity of non-peptidic ligands such as their cell internalization, trafficking within intracellular organelles, association with dedicated antigen-presenting molecules, induction of central and peripheral tolerance, and finally their role in autoimmune diseases as well as in protection during infections are unknown to date. The aims of this thesis were to assess some of the immunological functions and cell biological rules governing the immunogenicity of non-peptidic antigens, with particular emphasis on cell trafficking of non-peptidic antigens and antigen-presenting molecules. It focused on (i) the antigen reactivity and presence of human invariant natural killer T (iNKT) cells in diseases, (ii) the role of CD1a trafficking in lipid antigen presentation by this protein, and (iii) the requirements of membrane translocation of phosphorylated mevalonate metabolites that stimulate human T cell receptor (TCR) gamma-delta cells. With the development of alpha-galactosylceramide (alpha-GC)-loaded soluble CD1d dimers, which specifically interact with the TCR of iNKT cells, we have the perfect tool in our hands to perform detailed studies on iNKT cells. Analysis of the iNKT cells in blood unveiled large differences in their fluorescence intensity suggesting the presence of semi-invariant iNKT TCR with large disparities in the affinity for the alpha-GC-CD1d complex. Unexpectedly, established iNKT cell clones showed no correlation between CD1d dimer-staining levels and alpha-GC reactivity, indicating that additional mechanisms control responsiveness of iNKT cells, at least to this lipid antigen. The identification of lipid antigens stimulating exclusively some desired functions in human iNKT cells might lead to new medical therapies or vaccines. To screen a variety of synthetic lipids for their capacity to activate iNKT cells, we devised an in vitro model based on plastic-bound CD1d. Piperidinones, molecules with a ceramide- or sphingosine-like structure, revealed that a single lipid tail is sufficient to form stimulatory complexes with CD1d. Interestingly, piperidinones preferentially induce TH1-like cytokines, predicting a possible role as novel leader molecules to functionally direct iNKT cell responses deployable in clinical therapies. The balance of proinflammatory TH1 to regulatory TH2 cytokines is well-known to be decisive for the outcome of many diseases. Atherosclerosis (ATH) is a chronic inflammatory disease characterized by lipid accumulation in plaques. The disease is complicated by cardiovascular events provoked by plaque rupture or erosion. Because inflammation participates in lesion progression and rupture of plaques, the identification of its causes and of the culprit leukocyte populations involved in plaque destabilization is crucial for effective prevention of cardiovascular events. We used CD1d dimers to detect and characterize iNKT cells in ATH patients. We found that, in human atherosclerotic lesions, the abundance of CD1d-positive antigen-presenting cells (APC) and of iNKT cells correlates with disease severity and activity. CD1d-positive cells colonize advanced plaques in symptomatic patients and are most abundant in plaques with concomitant signs of ectopic neovascularization. In plaques, the frequency of iNKT cells among total T cells exceeds the one in blood. After having successfully isolated iNKT cell lines from plaque tissue, we showed that they promptly release proinflammatory cytokines upon lipid antigen stimulation and promote endothelial cell migration and microvascular sprout formation in vitro. This functional proangiogenic activity is ascribed to interleukin-8 released by iNKT cells after lipid recognition. These findings introduce iNKT cells as novel candidates to induce plaque neovascularization and destabilization in human ATH. Targeting iNKT cells could lead to late stage ATH treatment. Another approach to understand the role of lipid-specific immune responses is to investigate the molecular rules of lipid-CD1 complex formation. Lipids distribute, due to their physicochemical properties or with the help of specific transporters and lipid transfer proteins, to different intracellular compartments and membrane domains. Thus, it is advantageous for the immune system to utilize multiple CD1 isoforms, each with a distinct trafficking pattern, to facilitate sampling of lipid antigens localized in various membranes. Several studies have addressed trafficking of CD1 isoforms. However, the molecular mechanisms are known in only a few cases. We identified invariant chain (Ii) and lipid rafts as key regulators of CD1a organization on the surface of APC and of its immunological function as antigen-presenting molecule. Colocalization of CD1a with Ii is dependent on raft integrity and CD1a internalization is increased by Ii. The localization of CD1a in lipid rafts is functionally relevant as raft disruption inhibits CD1a-restricted antigen presentation. Moreover, we found that CD1a is internalized independently of clathrin and dynamin and that it follows a Rab22a- and adenosine diphosphate ribosylation factor (ARF) 6-dependent recycling pathway, similar to other clathrin-independent cargo. Posttranslational S-acylation of the CD1a cytoplasmic tail may occur but neither determines the rate of internalization nor recycling nor its localization to detergent-resistant membrane microdomains. These findings place CD1a close to major histocompatibility complex (MHC) class I in its trafficking routes although CD1a loads lipids in recycling endosomes and not in the endoplasmic reticulum as MHC class I. Strikingly, the glycolipid antigen sulfatide was found localized predominantly to early and recycling endosomes where CD1a is located. Swapping the cytoplasmic tail of CD1a for the one of CD1b and hence targeting the CD1a protein to the late endosomal and lysosomal compartments decreases its capacity to present sulfatide and shortens the half-life of stimulatory complexes. Thus, the physiological intracellular trafficking route of CD1a is critical for efficient presentation of lipid antigens that traffic through the early endocytic and recycling pathways. Intracellular trafficking of another class of non-peptidic antigens, namely the phosphorylated metabolites which stimulate human TCR gamma-delta cells expressing the Vgamma9/Vdelta2 heterodimer, was examined. These T cells recognize a family of structurally related compounds produced in the eukaryotic mevalonate and prokaryotic methylerythritol phosphate (MEP) pathways. The endogenous self-ligands are generated within the cytoplasm and must cross the membrane in order to associate with dedicated antigen-presenting molecules, which remain unknown at present. Using an in vitro transport assay, we demonstrated that the multidrug resistance-associated protein (MRP) 5 transporter is involved in membrane translocation of antigenic phosphorylated metabolites. Confocal microscopy illustrated that MRP5 is located in membranes of both endoplasmic reticulum and early endosomes. Both the intracellular localization and active role in antigen transport confer an immunological function to MRP5, resembling that of TAP (transporter associated with antigen processing) transporters involved in peptide antigen translocation. This indicates a similar strategy used for antigen presentation to TCR alpha-beta and gamma-delta T cells. In conclusion, these studies have underlined the physiological relevance of T cells recognizing non-peptidic ligands and have revealed unanticipated molecular mechanisms controlling the efficient presentation of such antigens

Commissioning ATLAS and CMS with top quarks

The large ttbar production cross-section at the LHC suggests the use of top quark decays to calibrate several critical parts of the detectors, such as the trigger system, the jet energy scale and b-tagging.Comment: 6 pages, 5 figures. Talk given at `V Workshop Italiano sulla Fisica pp a LHC', Perugia, Italy, 30 January - 2 February 200

Clearance of human papillomavirus related anal condylomas after oral and endorectal multistrain probiotic supplementation in an HIV positive male: A case report.

Abstract Go to: Introduction: Here we present the case of a 56-year-old human immunodeficiency virus (HIV)-infected man with multiple anal condylomas and positivity for human papilloma virus (HPV) 18 on anal brushing. Biopsies of the anal mucosa led to the diagnosis of Bowen's disease and a subsequent pelvic magnetic resonance imaging (MRI) scan evidenced multiple reactive lymphoadenopathies and large intra-anal condylomas. The patient was treated with a complete excision of Bowen's lesion and with a 4 months course of supplementation with a high concentration multistrain probiotic formulation administered orally and by rectal instillation with the purpose to reduce local inflammation and to enhance local mucosal immunity. Go to: Conclusion: An MRI performed at the end of the supplementation period evidenced the clearance of the anal condylomas previously described and no evidence of residual lymphadenopathies. Trials are therefore required to confirm this therapeutic possibility and for a better understanding of the mechanisms by which this specific probiotic formulation interacts with local epithelium when administered by the anal route

Anticipatory Postural Adjustments associated with reaching movements are programmed according to the availability of visual information

During goal-directed arm movements, the eyes, head, and arm are coordinated to look at and reach the target. We examined whether the expectancy of visual information about the target modifies Anticipatory Postural Adjustments (APAs). Ten standing subjects had to (1) move the eyes, head and arm, so as to reach, with both gaze and index-finger, a target of known position placed outside their visual field (Gaze-Reach); (2) look at the target while reaching it (Reach in Full Vision); (3) keep the gaze away until having touched it (Reach then Gaze) and (4) just Gaze without Reach the target. We recorded eye, head, right arm, and acromion kinematics, EMGs from upper- and lower-limb muscles, and forces exerted on the ground. In Gaze-Reach, two coordination strategies were found: when gaze preceded arm muscle recruitment (Gaze-first) and when the opposite occurred (Reach-first). APAs in acromion kinematics, leg muscles, and ground forces started significantly earlier in Gaze-first vs. Reach-first (mean time advance: 44.3 \ub1 8.9 ms), as it was in Reach in Full Vision vs. Reach then Gaze (39.5 \ub1 7.9 ms). The Gaze-first to Reach-first time-shift was similar to that between Reach in Full Vision and Reach then Gaze (p = 0.58). Moreover, Gaze without Reach data witnessed that the head-induced postural actions did not affect the APA onset in Gaze-first and Reach-first. In conclusion, in Gaze-first, the central control of posture considers visual information while planning the movement, like in Reach in Full Vision; while Reach-first is more similar to Reach then Gaze, where vision is not required

Intended rather than actual movement velocity determines the latency of anticipatory postural adjustments

The literature reports that anticipatory postural adjustments (APAs) are programmed according to movement velocity. However, the linkage between APAs and velocity has been highlighted within single subjects who were asked to voluntarily change movement velocity; therefore, till now, it has been impossible to discern whether the key factor determining APA latency was the intended movement velocity or the actual one. Aim of this study was to distinguish between these two factors. We analyzed the APA chain that stabilizes the arm during a brisk index finger flexion in two groups of subjects: (1) 29 who composed our database from previous experiments and were asked to "go-as-fast-as-possible" (go-fast), but actually performed the movement with different speeds (238-1180\ub0/s), and (2) ten new subjects who performed the go-fast movement at more than 500\ub0/s and were then asked to go-slow at about 50 % of their initial velocity, thus moving at 300-800\ub0/s. No correlation between APA latency and actual movement speed was observed when all subjects had to go-fast (p > 0.50), while delayed APAs were found in the ten new subjects when they had to go-slow (p < 0.001). Moreover, in the speed range between 300 and 800\ub0/s, the APA latency depended only on movement instruction: subjects going fast showed earlier APAs than those going slow (p < 0.001). These data suggest a stronger role of the intended movement velocity versus the actual one in modifying the timing of postural muscles recruitment with respect to the prime mover. These results also strengthen the idea of a shared postural and voluntary command within the same motor act

Performance of the Pwo Crystals of the Cms Electromagnetic Calorimeter

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Precision of a pointing movement performed with either the dominant or non-dominant hand is linked to the timing of anticipatory postural adjustments

Introduction: It is a common experience to feel motor awkwardness when performing a pointing movement with the non-preferred limb, which is known to be associated to less precise movements. Here we provide evidence that this last behaviour partly stems from changes in the temporal organization of the Anticipatory Postural Adjustments (APAs) in the non-preferred side. Materials and methods: We investigated the effect of lateralization on APAs in Biceps Brachii, Triceps Brachii and Anterior Deltoid, which stabilize the arm when performing a pen-pointing movement (prime mover Flexor Carpi Radialis). Moreover, we analysed the elbow and wrist kinematics as well as the precision of the pointing movement. Results: The mean kinematics of wrist movement and its latency, with respect to prime mover recruitment, were similar in the two sides, while APAs in Triceps Brachii, Biceps Brachii and Anterior Deltoid were less anticipated when movements were performed with the non-dominant (20\u201330 ms) versus dominant hand (60\u201370 ms). APAs in the non-dominant limb were associated with an altered fixation of the elbow, which showed a higher excursion, and with a more scattered pointing error (non-dominant: 16.3 \ub1 1.7 mm versus dominant: 10.1 \ub1 0.8 mm). Discussion: By securing the dynamics of the more proximal joints, an appropriate timing of the intra-limb APAs seems necessary for refining the voluntary movement precision. The linkage between APAs, elbow fixation and movement accuracy also agrees with the recent suggestion that APAs and prime mover recruitment are driven by a shared motor command, which strives to obtain an accurate pointing

Review article: liver transplantation for HCC. Treatment options on the waiting list

The most widely adopted criteria to admit and maintain patients with HCC and cirrhosis in the waiting list for liver transplantation are the Milano criteria, consisting in the presence of a single tumour ≤ 5 cm in diameter or up to three tumours, none exceeding 3 cm in diameter. Since the average time to transplantation has become longer than 10-12 months in most European and American Centers, the exclusion from the list during the waiting period due to increase of the neoplasm over the established criteria is not uncommon at present. It is mandatory, therefore, to seek an effective therapeutic strategy for patients with HCC waiting for transplantation. Surgical resection and eventual subsequent salvage transplantation seems a cost-effective strategy in resectable HCC. In unresectable neoplasms both transarterial chemoembolization and percutaneous ablation techniques are currently used and one or the other are chosen according to individual applicability, limitations and specific risks. However, although positive trends were reported, no definitive evidence has been produced so far about their efficacy in increasing patient's survival and decreasing tumour recurrence rates after transplantation. Adult-to-adult living donor liver transplantation is one possible way to shorten the waiting list, but this strategy involves important ethical implications. At present it appears justified to take it into consideration only if the waiting time for cadaveric OLT is expected to exceed 7 months. A more general and definitive attempt to overcome problems related to long waiting times for patients with HCC and relatively preserved hepatic function has been introduced in the USA very recently and consists in prioritizing patients with HCC. However, the overall efficacy of this approach will be established only in some years

Transcranial direct current stimulation of SMA modulates anticipatory postural adjustments without affecting the primary movement

Recent works provide evidences that anticipatory postural adjustments (APAs) are programmed with the prime mover recruitment as a shared posturo-focal command. However the ability of the CNS to adjust APAs to changes in the postural context implies that the postural and voluntary components should take different pathways before reaching the representation of single muscles in the primary motor cortex. Here we test if such bifurcation takes place at the level of the supplementary motor area (SMA). TDCS was applied over the SMA in 14 subjects, who produced a brisk index-finger flexion. This activity is preceded by inhibitory APAs, carved in the tonic activity of Biceps Brachii and Anterior Deltoid, and by an excitatory APA in Triceps Brachii. Subjects performed a series of 30 flexions before, during and after 20min of tDCS in CATHODAL, ANODAL or SHAM configuration. The inhibitory APA in Biceps and the excitatory APA in Triceps were both greater in ANODAL than in SHAM and CATHODAL configurations, while no difference was found among the latter two (ANODAL vs. SHAM: biceps +26.5%, triceps +66%; ANODAL vs. CATHODAL: biceps +20.5%, triceps: +63.4%; for both muscles, ANOVA p<0.02, Tukey p<0.05). Instead, the APA in anterior deltoid was unchanged in all configurations. No changes were observed in prime mover recruitment and index-finger kinematics. Results show that the SMA is involved in modulating APAs amplitude. Moreover, the differential effect of tDCS observed on postural and voluntary commands suggests that these two components of the motor program are already separated before entering SMA