Wiskott-Aldrich syndrome protein interacts and inhibits diacylglycerol kinase alpha promoting IL-2 induction

Background: Phosphorylation of diacylglycerol by diacylglycerol-kinases represents a major inhibitory event constraining T cell activation upon antigen engagement. Efficient TCR signalling requires the inhibition of the alpha isoform of diacylglycerol kinase, DGKα, by an unidentified signalling pathway triggered by the protein adaptor SAP. We previously demonstrated that, in SAP absence, excessive DGKα activity makes the T cells resistant to restimulation-induced cell death (RICD), an apoptotic program counteracting excessive T cell clonal expansion. Results: Herein, we report that the Wiskott-Aldrich syndrome protein (WASp) inhibits DGKα through a specific interaction of the DGKα recoverin homology domain with the WH1 domain of WASp. Indeed, WASp is necessary and sufficient for DGKα inhibition, and this WASp function is independent of ARP2/3 activity. The adaptor protein NCK-1 and the small G protein CDC42 connect WASp-mediated DGKα inhibition to SAP and the TCR signalosome. In primary human T cells, this new signalling pathway is necessary for a full response in terms of IL-2 production, while minimally affecting TCR signalling and restimulation-induced cell death. Conversely, in T cells made resistant to RICD by SAP silencing, the enhanced DAG signalling due to DGKα inhibition is sufficient to restore apoptosis sensitivity. Conclusion: We discover a novel signalling pathway where, upon strong TCR activation, the complex between WASp and DGKα blocks DGKα activity, allowing a full cytokine response

Identification of a novel DGKa inhibitor for XLP-1 therapy by virtual screening

As part of an effort to identify druggable diacylglycerol kinase alpha (DGKa) inhibitors, we used an insilico approach based on chemical homology with the two commercially available DGKa inhibitors R59022 and R59949. Ritanserin and compound AMB639752 emerged from the screening of 127 compounds, showing an inhibitory activity superior to the two commercial inhibitors, being furthermore specific for the alpha isoform of diacylglycerol kinase. Interestingly, AMB639752 was also devoid of serotoninergic activity. The ability of both ritanserin and AMB639752, by inhibiting DGKa in intact cells, to restore restimulation induced cell death (RICD) in SAP deficient lymphocytes was also tested. Both compounds restored RICD at concentrations lower than the two previously available inhibitors, indicating their potential use for the treatment of X-linked lymphoproliferative disease 1 (XLP-1), a rare genetic disorder in which DGKa activity is deregulated

The diacylglycerol kinase α/Atypical PKC/β1 integrin pathway in SDF-1α mammary carcinoma invasiveness

Diacylglycerol kinase α (DGKα), by phosphorylating diacylglycerol into phosphatidic acid, provides a key signal driving cell migration and matrix invasion. We previously demonstrated that in epithelial cells activation of DGKα activity promotes cytoskeletal remodeling and matrix invasion by recruiting atypical PKC at ruffling sites and by promoting RCP-mediated recycling of α5β1 integrin to the tip of pseudopods. In here we investigate the signaling pathway by which DGKα mediates SDF-1α-induced matrix invasion of MDA-MB-231 invasive breast carcinoma cells. Indeed we showed that, following SDF-1α stimulation, DGKα is activated and localized at cell protrusion, thus promoting their elongation and mediating SDF-1α induced MMP-9 metalloproteinase secretion and matrix invasion. Phosphatidic acid generated by DGKα promotes localization at cell protrusions of atypical PKCs which play an essential role downstream of DGKα by promoting Rac-mediated protrusion elongation and localized recruitment of β1 integrin and MMP-9. We finally demonstrate that activation of DGKα, atypical PKCs signaling and β1 integrin are all essential for MDA-MB-231 invasiveness. These data indicates the existence of a SDF-1α induced DGKα - atypical PKC - β1 integrin signaling pathway, which is essential for matrix invasion of carcinoma cells

Wiskott-Aldrich syndrome protein interacts and inhibits diacylglycerol kinase alpha promoting IL-2 induction

BackgroundPhosphorylation of diacylglycerol by diacylglycerol-kinases represents a major inhibitory event constraining T cell activation upon antigen engagement. Efficient TCR signalling requires the inhibition of the alpha isoform of diacylglycerol kinase, DGKα, by an unidentified signalling pathway triggered by the protein adaptor SAP. We previously demonstrated that, in SAP absence, excessive DGKα activity makes the T cells resistant to restimulation-induced cell death (RICD), an apoptotic program counteracting excessive T cell clonal expansion.ResultsHerein, we report that the Wiskott-Aldrich syndrome protein (WASp) inhibits DGKα through a specific interaction of the DGKα recoverin homology domain with the WH1 domain of WASp. Indeed, WASp is necessary and sufficient for DGKα inhibition, and this WASp function is independent of ARP2/3 activity. The adaptor protein NCK-1 and the small G protein CDC42 connect WASp-mediated DGKα inhibition to SAP and the TCR signalosome. In primary human T cells, this new signalling pathway is necessary for a full response in terms of IL-2 production, while minimally affecting TCR signalling and restimulation-induced cell death. Conversely, in T cells made resistant to RICD by SAP silencing, the enhanced DAG signalling due to DGKα inhibition is sufficient to restore apoptosis sensitivity.ConclusionWe discover a novel signalling pathway where, upon strong TCR activation, the complex between WASp and DGKα blocks DGKα activity, allowing a full cytokine response

Bowel preparation for elective colorectal resection: multi-treatment machine learning analysis on 6241 cases from a prospective Italian cohort

background current evidence concerning bowel preparation before elective colorectal surgery is still controversial. this study aimed to compare the incidence of anastomotic leakage (AL), surgical site infections (SSIs), and overall morbidity (any adverse event, OM) after elective colorectal surgery using four different types of bowel preparation. methods a prospective database gathered among 78 Italian surgical centers in two prospective studies, including 6241 patients who underwent elective colorectal resection with anastomosis for malignant or benign disease, was re-analyzed through a multi-treatment machine-learning model considering no bowel preparation (NBP; No. = 3742; 60.0%) as the reference treatment arm, compared to oral antibiotics alone (oA; No. = 406; 6.5%), mechanical bowel preparation alone (MBP; No. = 1486; 23.8%), or in combination with oAB (MoABP; No. = 607; 9.7%). twenty covariates related to biometric data, surgical procedures, perioperative management, and hospital/center data potentially affecting outcomes were included and balanced into the model. the primary endpoints were AL, SSIs, and OM. all the results were reported as odds ratio (OR) with 95% confidence intervals (95% CI). results compared to NBP, MBP showed significantly higher AL risk (OR 1.82; 95% CI 1.23-2.71; p = .003) and OM risk (OR 1.38; 95% CI 1.10-1.72; p = .005), no significant differences for all the endpoints were recorded in the oA group, whereas MoABP showed a significantly reduced SSI risk (OR 0.45; 95% CI 0.25-0.79; p = .008). conclusions MoABP significantly reduced the SSI risk after elective colorectal surgery, therefore representing a valid alternative to NBP

Abdominal drainage after elective colorectal surgery: propensity score-matched retrospective analysis of an Italian cohort

background: In italy, surgeons continue to drain the abdominal cavity in more than 50 per cent of patients after colorectal resection. the aim of this study was to evaluate the impact of abdominal drain placement on early adverse events in patients undergoing elective colorectal surgery. methods: a database was retrospectively analysed through a 1:1 propensity score-matching model including 21 covariates. the primary endpoint was the postoperative duration of stay, and the secondary endpoints were surgical site infections, infectious morbidity rate defined as surgical site infections plus pulmonary infections plus urinary infections, anastomotic leakage, overall morbidity rate, major morbidity rate, reoperation and mortality rates. the results of multiple logistic regression analyses were presented as odds ratios (OR) and 95 per cent c.i. results: a total of 6157 patients were analysed to produce two well-balanced groups of 1802 patients: group (A), no abdominal drain(s) and group (B), abdominal drain(s). group a versus group B showed a significantly lower risk of postoperative duration of stay >6 days (OR 0.60; 95 per cent c.i. 0.51-0.70; P < 0.001). a mean postoperative duration of stay difference of 0.86 days was detected between groups. no difference was recorded between the two groups for all the other endpoints. conclusion: this study confirms that placement of abdominal drain(s) after elective colorectal surgery is associated with a non-clinically significant longer (0.86 days) postoperative duration of stay but has no impact on any other secondary outcomes, confirming that abdominal drains should not be used routinely in colorectal surgery

Regulation of Diacylglycerol Kinase alpha in controlling activated T cell fate

Le cellule T CD8+ sono designate per individuare ed eliminare patogeni intracellulari (ad esempio i virus), creando un pool di cellule T memoria per un migliore controllo a successive infezioni. Durante la risposta immunitaria sia primaria che secondaria, le cellule T effettrici attivate proliferano rapidamente per controllare ed eliminare un patogeno invasivo. Tuttavia l\u2019espansione delle cellule CD8+ deve essere bilanciata ad una morte cellulare programmata per prevenire danni collaterali all\u2019ospite. La sindrome linfoproliferativa legata al cromosoma X (X-linked lymphoproliferative disease) XLP-1 \ue8 un\u2019immunodeficienza primaria associata ad un\u2019eccessiva espansione delle cellule T CD8+ in seguito all\u2019infezione del virus Epstein-Barr (EBV). XLP-1 \ue8 causata da un difetto genetico di SAP, una proteina adattatrice che facilita il signaling attraverso i recettori della famiglia SLAM. Cellule prive di SAP mostrano un\u2019alterata morte indotta da restimolazione (restimulation-induced cell death; RICD), attraverso il T cell receptor (TCR), un processo che normalmente limita l\u2019espansione delle cellule T durante la risposta immunitaria per precludere un massivo accumulo delle cellule T stesse. Cellule prive di SAP mostrano, inoltre, un difetto di inibizione della diacilglicerolo (DAG) chinasi alfa (DGK\u3b1), un enzima che converte il DAG ad acido fosfatidico per modulare il signaling del TCR. Elevati livelli di attivit\ue0 di DGK\u3b1 portano ad un aumento del turnover di DAG e ad una diminuzione dell\u2019attivazione di RasGRP and PKC\u3b8 in seguito alla stimolazione del TCR. Il primo obiettivo di questo lavoro \ue8 stato di valutare se e come un\u2019aumentata attivit\ue0 funzionale di DGK\u3b1 possa contribuire alla resistenza alla RICD nelle cellule T di pazienti XLP-1. Abbiamo quindi dimostrato che il silenziamento mediante l\u2019utilizzo di specifici siRNA o l\u2019inibizione farmacologica di DGK\u3b1 promuove RICD in cellule T prive SAP. Il silenziamento/ inibizione farmacologica di DGK\u3b1 \ue8 sufficiente a recuperare il difetto della polarizzazione di DAG, la formazione della sinapsi immunologica e il reclutamento di PKC\u3b8/RasGRP alla sinapsi immunologica delle cellule T prive di SAP. Sorprendentemente, abbiamo scoperto che l\u2019inibizione di DGK\u3b1 specificamente recupera l\u2019espressione delle proteine pro-apoptotiche indotte dal TCR quali NUR77 and NOR1, che ripristinano la sensibilit\ue0 alla RICD delle cellule T prive di SAP. In vivo, l\u2019inibizione farmacologica di DGK\u3b1 previene l\u2019eccessiva espansione delle cellule T CD8+ e la produzione di IFN-\u3b3 in topi SAP\u2013\u2044\u2013 infettati con il virus Lymphocytic Choriomeningitis (LCMV), mettendo in evidenza che DGK\u3b1 possa essere target terapeutico attraente per invertire l\u2019immunopatologia associata ad EBV osservata nei pazienti XLP-1 correggendo RICD. Per meglio poter delucidare il signaling pathway grazie al quale la restimolazione del TCR possa indurre l\u2019inibizione di DGK\u3b1 attraverso l\u2019attivazione o il reclutamento di diverse proteine, abbiamo voluto studiare il coinvolgimento degli interattori di SAP. Abbiamo quindi dimostrato che SAP inibisce l\u2019attivit\ue0 di DGK\u3b1 mediante un meccanismo che richiede NCK1, CDC42, WASp e Arp2/3, suggerendo che la polimerizzazione dell\u2019actina sia necessaria per RICD in maniera SAP dipendente. Il rimodellamento del citoscheletro \ue8 importante sia per l\u2019attivazione che l\u2019interazione con le cellule ed il microambiente, ed esso \ue8 in parte governato dalle integrine. Per valutare la possibilit\ue0 che l\u2019adesione mediante integrine svolga un ruolo prominente nel pathway SAP-DGK\u3b1, e sapendo che \ue8 presente un link funzionale tra \u3b21 integrin e DGK\u3b1, ci siamo chiesti se \u3b21 integrin possa partecipare alla RICD. Sorprendentemente le cellule T silenziate per \u3b21 integrin presentano una significativa riduzione nella RICD, che viene completamente recuperata dal silenziamento di DGK\u3b1. Inoltre, abbiamo osservato un\u2019aumentata RICD quando \u3b21 integrin viene simultaneamente reclutata con il TCR suggerendo che la stimolazione attraverso il TCR, porta ad una inibizione di DGK\u3b1 attraverso SAP che prevede anche il coinvolgimento di \u3b21 integrin e il rimodellamento dell\u2019actina alla sinapsi immunologica. Il signaling di \u3b21 integrin probabilmente gioca un ruolo prominente nel potenziare RICD come co-recettore che aiuta il signaling del TCR per raggiungere la soglia richiesta per indurre la morte cellulare. Riassumendo questo lavoro mette in evidenzia una nuova via di segnalazione che collega SAP, DGK\u3b1, \u3b21 integrin e le proteine coinvolte nel rimodellamento actinico, ad un programma autoregolatorio critico che porta all\u2019apoptosi nelle cellule T

Clinicopathological evaluation of 59 cases of fetal death

OBJECTIVE: The purpose of this study has been assessing the determinants of stillbirth among the newborns of the Verona University Obstetrics Department. MATERIALS AND METHODS: A total of 59 stillbirth cases, observed between January 2000 and June 2006, were retrospectively studied. WHO definition for stillbirth was adopted as the inclusion criterion. Clinical files, feto-maternal laboratory data, feto-placental pathology findings as well as delivery mode and circumstances were all systematically reviewed. RESULTS: The 59 observed cases correspond to an incidence of 9.8 stillbirths/year, which, considering the institutional delivery rate, correspond to 5.4 cases per 1000 births. Frequent relevant conditions associated with stillbirth were intrauterine growth restriction (15.2%), congenital fetal anomalies (13.5%), various maternal diseases (21.0%); no cause of fetal demise could be found in 10/59 (17.0%) cases, which were classified as unexplained. Most deliveries were successfully induced with prostaglandins except 11 cases (19.0%) which required a C-section due to severe maternal conditions associated with the fetal loss. CONCLUSION: Thorough investigation of each individual stillbirth case, by means of an integrated study protocol, along with the Pathologist's close collaboration, allows identification of a likely cause in the majority of cases. Better knowledge of unexpected fetal loss is the premise for better parental counselling and for prevention of recurrences