Co-recruitment analysis of the CBL and CBLB signalosomes in primary T cells identifies CD5 as a key regulator of TCR-induced ubiquitylation.

T-cell receptor (TCR) signaling is essential for the function of T cells and negatively regulated by the E3 ubiquitin-protein ligases CBL and CBLB Here, we combined mouse genetics and affinity purification coupled to quantitative mass spectrometry to monitor the dynamics of the CBL and CBLB signaling complexes that assemble in normal T cells over 600 seconds of TCR stimulation. We identify most previously known CBL and CBLB interacting partners, as well as a majority of proteins that have not yet been implicated in those signaling complexes. We exploit correlations in protein association with CBL and CBLB as a function of time of TCR stimulation for predicting the occurrence of direct physical association between them. By combining co-recruitment analysis with biochemical analysis, we demonstrated that the CD5 transmembrane receptor constitutes a key scaffold for CBL- and CBLB-mediated ubiquitylation following TCR engagement. Our results offer an integrated view of the CBL and CBLB signaling complexes induced by TCR stimulation and provide a molecular basis for their negative regulatory function in normal T cells.

The membrane adaptor LAT is proteolytically cleaved following Fas engagement in a tyrosine phosphorylation-dependent fashion.

Engagement of the TCR (T-cell receptor) induces tyrosine phosphorylation of the LAT (linker for the activation of T-cells) adaptor, and thereby it recruits several cytosolic mediators for downstream signalling pathways. The Fas protein is essential for T-lymphocyte apoptosis, and following Fas engagement, many proteins are proteolytically cleaved, including several molecules that are important for the transduction of TCR intracellular signals. In the present study, we demonstrate that the adaptor LAT is also subject to a proteolytic cleavage in mature T-lymphocytes and thymocytes in response to Fas engagement, and also on TCR stimulation, and we identify three aspartic acid residues at which LAT is cleaved. Interestingly, these aspartic acid residues are located in proximity to several functionally important tyrosine residues of LAT, raising the possibility that their phosphorylation could modulate LAT cleavage. Consistent with that hypothesis, we show that induction of phosphorylation by pervanadate or H2O2 in Jurkat cells and thymocytes inhibits Fas-mediated cleavage of LAT. Moreover, we show that LAT proteolysis is also enhanced during anergy induction of primary human T-cells, suggesting that LAT cleavage may act as a regulator of TCR-mediated activation of T-cells and not only as a transducer of cell death promoting stimuli.

The effect of L-alanyl-L-glutamine dipeptide supplemented total parenteral nutrition on infectious morbidity and insulin sensitivity in critically ill patients.

OBJECTIVE: The aim of this study was to assess the clinical efficacy of alanine-glutamine dipeptide-supplemented total parenteral nutrition defined by the occurrence of nosocomial infections. Secondary parameters included Sequential Organ Failure Assessment score, hyperglycemia and insulin needs, intensive care unit and hospital length of stay, and 6-month mortality. DESIGN: Multicenter, prospective, double-blind, randomized trial. SETTING: Twelve intensive care units at Spanish hospitals. PATIENTS: One hundred twenty-seven patients with Acute Physiology and Chronic Health Evaluation II score >12 and requiring parenteral nutrition for 5-9 days. INTERVENTION: Patients were randomized to receive an isonitrogenous and isocaloric total parenteral nutrition or alanine-glutamine dipeptide-supplemented total parenteral nutrition. Nutritional needs were calculated: 0.25 g N/kg(-1)/d(-1) and 25 kcal/kg(-1)/d(-1). The study group received 0.5 g/kg(-1)/d(-1) of glutamine dipeptide and the control total parenteral nutrition group a similar amount of amino acids. Hyperglycemia was controlled applying an intensive insulin protocol with a target glycemia of 140 mg/dL. MEASUREMENTS AND MAIN RESULTS: The two groups did not differ at inclusion for the type and severity of injury or the presence of sepsis or septic shock. Caloric intake was similar in both groups. Preprotocol analysis showed that treated patients with alanine-glutamine dipeptide-supplemented total parenteral nutrition had lesser nosocomial pneumonia, 8.04 vs. 29.25 episodes-‰ days of mechanical ventilation (p = .02), and urinary tract infections, 2.5 vs. 16.7 episodes-‰ days of urinary catheter (p = .04). Intensive care unit, hospital, and 6-month survival were not different. Mean plasmatic glycemia was 149 ± 46 mg/dL in the alanine-glutamine dipeptide-supplemented total parenteral nutrition group and 155 ± 51 mg/dL in the control total parenteral nutrition group (p < .04), and mean hourly insulin dose was 4.3 ± 3.3 IU in the alanine-glutamine dipeptide-supplemented total parenteral nutrition group and 4.7 ± 3.7 IU in control total parenteral nutrition group (p < .001). Multivariate analysis showed a 54% reduction of the amount of insulin for the same levels of glycemia in the alanine-glutamine dipeptide-supplemented total parenteral nutrition group. CONCLUSIONS: Total parenteral nutrition supplemented with alanine-glutamine in intensive care unit patients is associated with a reduced rate of infectious complications and better glycemic control.

Liver dysfunction associated with artificial nutrition in critically ill patients.

INTRODUCTION: Liver dysfunction associated with artificial nutrition in critically ill patients is a complication that seems to be frequent, but it has not been assessed previously in a large cohort of critically ill patients. METHODS: We conducted a prospective cohort study of incidence in 40 intensive care units. Different liver dysfunction patterns were defined: (a) cholestasis: alkaline phosphatase of more than 280 IU/l, gamma-glutamyl-transferase of more than 50 IU/l, or bilirubin of more than 1.2 mg/dl; (b) liver necrosis: aspartate aminotransferase of more than 40 IU/l or alanine aminotransferase of more than 42 IU/l, plus bilirubin of more than 1.2 mg/dl or international normalized ratio of more than 1.4; and (c) mixed pattern: alkaline phosphatase of more than 280 IU/l or gamma-glutamyl-transferase of more than 50 IU/l, plus aspartate aminotransferase of more than 40 IU/l or alanine aminotransferase of more than 42 IU/l. RESULTS: Seven hundred and twenty-five of 3,409 patients received artificial nutrition: 303 received total parenteral nutrition (TPN) and 422 received enteral nutrition (EN). Twenty-three percent of patients developed liver dysfunction: 30% in the TPN group and 18% in the EN group. The univariate analysis showed an association between liver dysfunction and TPN (p < 0.001), Multiple Organ Dysfunction Score on admission (p < 0.001), sepsis (p < 0.001), early use of artificial nutrition (p < 0.03), and malnutrition (p < 0.01). In the multivariate analysis, liver dysfunction was associated with TPN (p < 0.001), sepsis (p < 0.02), early use of artificial nutrition (p < 0.03), and calculated energy requirements of more than 25 kcal/kg per day (p < 0.05). CONCLUSION: TPN, sepsis, and excessive calculated energy requirements appear as risk factors for developing liver dysfunction. Septic critically ill patients should not be fed with excessive caloric amounts, particularly when TPN is employed. Administering artificial nutrition in the first 24 hours after admission seems to have a protective effect.

[Assessment of the clinical quality indicator "early enteral nutrition" in intensive care units]. / Evaluación del indicador clínico de calidad "nutrición enteral precoz" en servicios de medicina intensiva.

BACKGROUND: Early enteral nutrition (EN) offers multiple benefits on critically ill patients and its monitoring was established as a clinical quality indicator (CQI) for intensive care units (ICU) by the Spanish Society of Critical Care (SEMICYUC). However, no results have been published on the systematized monitoring of this CQI. OBJECTIVE: To assess the compliance of the CQI "Early EN" at ICU. METHODS: A retrospective longitudinal study was conducted on 386 consecutive ICU patients receiving EN. Data were collected including time from admission until EN was started, as well as clinical results. SPSS statistics software was used for analysis and the CQI was assessed according to SEMYCIUC criteria. RESULTS AND CONCLUSION: CQI's compliance was 70.2%, not reaching the settled standard of 100%. Barriers preventing CQI compliance ought to be analyzed and its standard of 100% reconsidered.

Precise Temporal Profiling of Signaling Complexes in Primary Cells Using SWATH Mass Spectrometry.

Spatiotemporal organization of protein interactions in cell signaling is a fundamental process that drives cellular functions. Given differential protein expression across tissues and developmental stages, the architecture and dynamics of signaling interaction proteomes is, likely, highly context dependent. However, current interaction information has been almost exclusively obtained from transformed cells. In this study, we applied an advanced and robust workflow combining mouse genetics and affinity purification (AP)-SWATH mass spectrometry to profile the dynamics of 53 high-confidence protein interactions in primary T cells, using the scaffold protein GRB2 as a model. The workflow also provided a sufficient level of robustness to pinpoint differential interaction dynamics between two similar, but functionally distinct, primary T cell populations. Altogether, we demonstrated that precise and reproducible quantitative measurements of protein interaction dynamics can be achieved in primary cells isolated from mammalian tissues, allowing resolution of the tissue-specific context of cell-signaling events.

Immunonutrition in the intensive care unit. A systematic review and consensus statement.

OBJECTIVE: To systematically review the effects of enteral nutrition with pharmaconutrients-enriched diets in critically ill patients and to establish recommendations for their use. DATA SOURCES: Computerized bibliographic search of published research and citation review of relevant articles. STUDY SELECTION: Randomized clinical trials of critically ill patients treated with enteral nutrition comparing diets enriched with pharmaconutrients vs not enriched diets were included. Infectious complications and outcome variables (days on mechanical ventilation, ICU and hospital length of stay and mortality) were evaluated. Studies were classified in four subgroups according to the patient's primary diagnosis: surgical, trauma, burned or medical. DATA EXTRACTION: A group of experts in methodology performed data extraction and statistical processes. A global analysis of the studies was done and also a separate study for each subgroup. Results of the meta-analysis were discussed within a 'clinical group' of clinicians with experience in the nutritional support of ICU patients, in order to find agreement about recommendations for the use of pharmaconutrients-enriched diets in critically ill patients. RESULTS: Independent review of 267 articles identified 26 relevant primary studies. Global results indicate that there was a reduction in infection rate in the pharmaconutrition group, considering the appreciated lower incidence in abdominal abscesses (OR: 0.26, CI: 0.12-0.55) (P=0.005), nosocomial pneumonia (OR: 0.54, CI: 0.35-0.84) (P=0.007) and bacteremia (OR: 0.45, CI: 0.35-0.84) (P=0.0002). Also, patients treated with pharmaconutrition diets have a reduction in time on mechanical ventilation (mean 2.25 days, CI: 0.5-3.9) (P=0.009), ICU length of stay (mean reduction of 1.6 days, CI: 1.9-1.2) (P<0.0001) and hospital length of stay (mean reduction of 3.4 days, CI: 4.0-2.7) (P<0.0001). No effects were appreciated on mortality (OR: 1.10, CI: 0.85-1.42) (P=0.5). Nevertheless, the separate analysis for each subgroup showed that the reported beneficial effects were not the same for each patient population. Also, the clinician panel of experts identifies several problems in the published data about enteral pharmaconutrition in critically ill patients. In spite of the subgroup differences and of the problems detected, the clinician group considered that the appreciated results could support a Grade B recommendation for the use of these formulas in ICU patients. CONCLUSIONS: Considering the beneficial effects and the absence of detrimental ones, the use of diets enriched with pharmaconutrients could be recommended in ICU patients requiring enteral feeding. Nevertheless, more investigation is needed in this field in order to find the more appropriate population of patients that can benefit from this nutritional therapy.

Evaluación del indicador clínico de calidad «nutrición enteral precoz» en servicios de medicina intensiva / Assessment of the clinical quality indicator «early enteral nutrition» in intensive care units

Introducción: la nutrición enteral (NE) precoz presenta múltiples beneficios en pacientes críticos y su monitorización ha sido establecida por la Sociedad Española de Medicina Intensiva, Crítica y Unidades Coronarias (SEMICYUC) como indicador clínico de calidad (ICC) para unidades de cuidados intensivos (UCI). Sin embargo, no se han publicado resultados sobre la monitorización reglada de dicho indicador. Objetivo: evaluar el cumplimiento del ICC NE precoz. Métodos: estudio longitudinal retrospectivo en 386 pacientes consecutivos de UCI que recibieron NE. Se recogieron variables básicas, número de horas transcurridas desde el ingreso hasta instaurarse la NE y resultado clínico de los pacientes. Para el análisis estadístico se empleó el software SPSS Statistics y el ICC fue evaluado mediante criterios de SEMICYUC. Resultados y conclusión: el cumplimiento del ICC fue del 70,2%, no alcanzando el estándar establecido (100%). Es necesario analizar las barreras que impiden el cumplimiento del estándar de este ICC y reconsiderar su valor del 100% (AU)

Background: Early enteral nutrition (EN) offers multiple benefits on critically ill patients and its monitoring was established as a clinical quality indicator (CQI) for intensive care units (ICU) by the Spanish Society of Critical Care (SEMICYUC). However, no results have been published on the systematized monitoring of this CQI. Objective: To assess the compliance of the CQI «Early EN» at ICU. Methods: A retrospective longitudinal study was conducted on 386 consecutive ICU patients receiving EN. Data were collected including time from admission until EN was started, as well as clinical results. SPSS statistics software was used for analysis and the CQI was assessed according to SEMYCIUC criteria. Results and conclusion: CQI's compliance was 70.2%, not reaching the settled standard of 100%. Barriers preventing CQI compliance ought to be analyzed and its standard of 100% reconsidered (AU)

Serine residues in the LAT adaptor are essential for TCR-dependent signal transduction.

The adaptor protein LAT has a prominent role in the transduction of intracellular signals elicited by the TCR/CD3 complex. Upon TCR engagement, LAT becomes tyrosine-phosphorylated and thereby, recruits to the membrane several proteins implicated in the activation of downstream signaling pathways. However, little is known about the role of other conserved motifs present in the LAT sequence. Here, we report that the adaptor LAT contains several conserved serine-based motifs, which are essential for proper signal transduction through the TCR. Mutation of these serine motifs in the human T cell line Jurkat prevents proper calcium influx, MAPK activation, and IL-2 production in response to TCR/CD3 stimulation. Moreover, this mutant form of LAT has a reduced ability to bind to PLC-γ1 and SLP-76, although phosphorylation of tyrosine residues 132, 171, and 191 is not decreased, raising a possible role for the serine-based motifs of LAT for the binding of important partners. The functional role of LAT serine-based motifs in signal transduction could be mediated by an effect on tyrosine phosphorylation, as their mutation significantly diminishes the phosphorylation of tyrosine residue 226. In addition, these serine motifs seem to have a regulatory role, given that upon their mutation, ZAP-70 shows enhanced phosphorylation. Therefore, the LAT serine-based motifs likely regulate signaling pathways that are essential for T cell physiology.