Role of fucosyltransferase-VII and pselectin glycoprotein ligant-1 in the suppressor activity of CD25+CD4+ regulatory T cells.

Ricerca sulla caratterizzazione dei meccanismi molecolari che controllano la migrazione e la capacità soppressoria dei linfociti CD4+CD25+ (cellule T regolatorie) durante l’encefalomielite sperimentale autoimmune.ope

An unexpected link between fatty acid synthase and cholesterol synthesis in proinflammatory macrophage activation

Different immune activation states require distinct metabolic features and activities in immune cells. For instance, inhibition of fatty acid synthase (FASN), which catalyzes the synthesis of long-chain fatty acids, prevents the proinflammatory response in macrophages; however, the precise role of this enzyme in this response remains poorly defined. Consistent with previous studies, we found here that FASN is essential for lipopolysaccharide-induced, Toll-like receptor (TLR)-mediated macrophage activation. Interestingly, only agents that block FASN upstream of acetoacetyl-CoA synthesis, including the well-characterized FASN inhibitor C75, inhibited TLR4 signaling, while those acting downstream had no effect. We found that acetoacetyl-CoA could overcome C75's inhibitory effect, whereas other FASN metabolites, including palmitate, did not prevent C75-mediated inhibition. This suggested an unexpected role for acetoacetyl-CoA in inflammation that is independent of its role in palmitate synthesis. Our evidence further suggested that acetoacetyl-CoA arising from FASN activity promotes cholesterol production, indicating a surprising link between fatty acid synthesis and cholesterol synthesis. We further demonstrate that this process is required for TLR4 to enter lipid rafts and facilitate TLR4 signaling. In conclusion, we have uncovered an unexpected link between FASN and cholesterol synthesis that appears to be required for TLR signal transduction and proinflammatory macrophage activation

Challenging the diagnosis of Cystic Fibrosis in a patient carrying the 186-8T/C allelic variant in the CF Transmembrane Conductance Regulator gene

BACKGROUND: This report describe for the first time a clinical case with a CFTR allelic variant 186-8T/C (c.54-8 T/C) in intron 1 of CFTR and underline the importance of applying a combination of genetic and functional tests to establish or exclude a diagnosis of Cystic Fibrosis. In this case the diagnostic algorithm proposed for CF has been successfully applied at our Center and previous CF diagnosis assigned in a different Center was not confirmed.Case report: A 38 year-old Italian woman had been treated as affected by CF since 2010, following diagnosis based on sweat tests (reported values of 73 and 57 mEq/L) and a clinical history consistent with CF. No mutations were identified by first level of genetic analysis. Afterwards the patient referred to our center for assessing the relevance of these findings. The genetic variant 186-8T/C (c.54-8 T/C) in intron 1 of the CFTR gene was detected by sequencing. Low-level interstitial-alveolar infiltration was recorded by high-resolution computerized tomography. Lung function was normal and sputum and Broncho Alveolar Lavage cultures resulted bacteriologically negative. Sweat chloride levels was re-assessed and resulted with values of 57 and 35 mEq/L, with a borderline range between 40 and 60 mEq/L. Nasal Potential Difference measurements resulted in three reliable measurements consistent with a non-CF phenotype. Differential diagnosis with ciliary dyskinesia was excluded, as was exon 2 skipping of CFTR gene that might have caused a CFTR functional defect. Furthermore, single cell fluorescence analysis in response to cAMP agonists performed in patient's monocytes overlapped those obtained in healthy donors. CONCLUSION: We concluded that this patient was not affected by CF. This case highlights the need for referrals to highly specialized centers and the importance of combined functional and genetic tests in making a correct diagnosis. Moreover, we confirmed a correlation between NPD tracings and cell depolarization in monocytes providing a rationale for proposing the use of leukocytes as a potential support for CF diagnosis

Mechanisms of T cell organotropism

F.M.M.-B. is supported by the British Heart Foundation, the Medical Research Council of the UK and the Gates Foundation

LFA-1 Controls Th1 and Th17 Motility Behavior in the Inflamed Central Nervous System

Leukocyte trafficking is a key event during autoimmune and inflammatory responses. The subarachnoid space (SAS) and cerebrospinal fluid are major routes for the migration of encephalitogenic T cells into the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis, and are sites of T cell activation before the invasion of CNS parenchyma. In particular, autoreactive Th1 and Th17 cell trafficking and reactivation in the CNS are required for the pathogenesis of EAE. However, the molecular mechanisms controlling T cell dynamics during EAE are unclear. We used two-photon laser microscopy to show that autoreactive Th1 and Th17 cells display distinct motility behavior within the SAS in the spinal cords of mice immunized with the myelin oligodendrocyte glycoprotein peptide MOG(35-55). Th1 cells showed a strong directional bias at the disease peak, moving in a straight line and covering long distances, whereas Th17 cells exhibited more constrained motility. The dynamics of both Th1 and Th17 cells were strongly affected by blocking the integrin LFA-1, which interfered with the deformability and biomechanics of Th1 but not Th17 cells. The intrathecal injection of a blocking anti-LFA-1 antibody at the onset of disease significantly inhibited EAE progression and also strongly reduced neuro-inflammation in the immunized mice. Our results show that LFA-1 plays a pivotal role in T cell motility during EAE and suggest that interfering with the molecular mechanisms controlling T cell motility can help to reduce the pathogenic potential of autoreactive lymphocytes

Challenging the diagnosis of Cystic Fibrosis in a patient carrying the 186-8T/C allelic variant in the CF Transmembrane Conductance Regulator gene

BACKGROUND: This report describe for the first time a clinical case with a CFTR allelic variant 186-8T/C (c.54-8 T/C) in intron 1 of CFTR and underline the importance of applying a combination of genetic and functional tests to establish or exclude a diagnosis of Cystic Fibrosis. In this case the diagnostic algorithm proposed for CF has been successfully applied at our Center and previous CF diagnosis assigned in a different Center was not confirmed.Case report: A 38 year-old Italian woman had been treated as affected by CF since 2010, following diagnosis based on sweat tests (reported values of 73 and 57 mEq/L) and a clinical history consistent with CF. No mutations were identified by first level of genetic analysis. Afterwards the patient referred to our center for assessing the relevance of these findings. The genetic variant 186-8T/C (c.54-8 T/C) in intron 1 of the CFTR gene was detected by sequencing. Low-level interstitial-alveolar infiltration was recorded by high-resolution computerized tomography. Lung function was normal and sputum and Broncho Alveolar Lavage cultures resulted bacteriologically negative. Sweat chloride levels was re-assessed and resulted with values of 57 and 35 mEq/L, with a borderline range between 40 and 60 mEq/L. Nasal Potential Difference measurements resulted in three reliable measurements consistent with a non-CF phenotype. Differential diagnosis with ciliary dyskinesia was excluded, as was exon 2 skipping of CFTR gene that might have caused a CFTR functional defect. Furthermore, single cell fluorescence analysis in response to cAMP agonists performed in patient's monocytes overlapped those obtained in healthy donors. CONCLUSION: We concluded that this patient was not affected by CF. This case highlights the need for referrals to highly specialized centers and the importance of combined functional and genetic tests in making a correct diagnosis. Moreover, we confirmed a correlation between NPD tracings and cell depolarization in monocytes providing a rationale for proposing the use of leukocytes as a potential support for CF diagnosis

Challenging the diagnosis of Cystic Fibrosis in a patient carrying the 186-8T/C allelic variant in the CF Transmembrane Conductance Regulator gene

BACKGROUND: This report describe for the first time a clinical case with a CFTR allelic variant 186-8T/C (c.54-8 T/C) in intron 1 of CFTR and underline the importance of applying a combination of genetic and functional tests to establish or exclude a diagnosis of Cystic Fibrosis. In this case the diagnostic algorithm proposed for CF has been successfully applied at our Center and previous CF diagnosis assigned in a different Center was not confirmed.Case report: A 38 year-old Italian woman had been treated as affected by CF since 2010, following diagnosis based on sweat tests (reported values of 73 and 57 mEq/L) and a clinical history consistent with CF. No mutations were identified by first level of genetic analysis. Afterwards the patient referred to our center for assessing the relevance of these findings. The genetic variant 186-8T/C (c.54-8 T/C) in intron 1 of the CFTR gene was detected by sequencing. Low-level interstitial-alveolar infiltration was recorded by high-resolution computerized tomography. Lung function was normal and sputum and Broncho Alveolar Lavage cultures resulted bacteriologically negative. Sweat chloride levels was re-assessed and resulted with values of 57 and 35 mEq/L, with a borderline range between 40 and 60 mEq/L. Nasal Potential Difference measurements resulted in three reliable measurements consistent with a non-CF phenotype. Differential diagnosis with ciliary dyskinesia was excluded, as was exon 2 skipping of CFTR gene that might have caused a CFTR functional defect. Furthermore, single cell fluorescence analysis in response to cAMP agonists performed in patient's monocytes overlapped those obtained in healthy donors. CONCLUSION: We concluded that this patient was not affected by CF. This case highlights the need for referrals to highly specialized centers and the importance of combined functional and genetic tests in making a correct diagnosis. Moreover, we confirmed a correlation between NPD tracings and cell depolarization in monocytes providing a rationale for proposing the use of leukocytes as a potential support for CF diagnosis

Coenzyme A fueling with pantethine limits autoreactive T cell pathogenicity in experimental neuroinflammation

Background: Immune cell metabolism governs the outcome of immune responses and contributes to the development of autoimmunity by controlling lymphocyte pathogenic potential. In this study, we evaluated the metabolic profile of myelin-specific murine encephalitogenic T cells, to identify novel therapeutic targets for autoimmune neuroinflammation. Methods: We performed metabolomics analysis on actively-proliferating encephalitogenic T cells to study their overall metabolic profile in comparison to resting T cells. Metabolomics, phosphoproteomics, in vitro functional assays, and in vivo studies in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS), were then implemented to evaluate the effect of metabolic targeting on autoreactive T cell pathogenicity. Finally, we confirmed the translational potential of our targeting approach in human pro-inflammatory T helper cell subsets and in T cells from MS patients. Results: We found that autoreactive encephalitogenic T cells display an altered coenzyme A (CoA) synthesis pathway, compared to resting T cells. CoA fueling with the CoA precursor pantethine (PTTH) affected essential immune-related processes of myelin-specific T cells, such as cell proliferation, cytokine production, and cell adhesion, both in vitro and in vivo. Accordingly, pre-clinical treatment with PTTH before disease onset inhibited the development of EAE by limiting T cell pro-inflammatory potential in vivo. Importantly, PTTH also significantly ameliorated the disease course when administered after disease onset in a therapeutic setting. Finally, PTTH reduced pro-inflammatory cytokine production by human T helper 1 (Th1) and Th17 cells and by T cells from MS patients, confirming its translational potential. Conclusion: Our data demonstrate that CoA fueling with PTTH in pro-inflammatory and autoreactive T cells may represent a novel therapeutic approach for the treatment of autoimmune neuroinflammation