Elisabet Cantó

Effect of diet composition and ration size on key enzyme activities of glycolysis–gluconeogenesis, the pentose phosphate pathway and amino acid metabolism in liver of gilthead sea bream ( Sparus aurata )

The effects of diet composition and ration size on the activities of key enzymes involved in intermediary metabolism were studied in the liver of gilthead sea bream (Sparus aurata). High-carbohydrate, low-protein diets stimulated 6-phosphofructo 1-kinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) enzyme activities, while they decreased alanine aminotransferase (EC 2.6.1.2) activity. A high degree of correlation was found between food ration size and the activity of the enzymes 6-phosphofructo 1-kinase, pyruvate kinase, glucose-6-phosphate dehydrogenase (positive correlations) and fructose-1,6-bisphosphatase (EC 3.1.3.11) (negative correlation). These correlations matched well with the high correlation also found between ration size and growth rate in starved fish refed for 22 d. Limited feeding (5 g/kg body weight) for 22 d decreased the activities of the key enzymes for glycolysis and lipogenesis, and alanine aminotransferase activity. The findings presented here indicate a high level of metabolic adaptation to both diet type and ration size. In particular, adaptation of enzyme activities to the consumption of a diet with a high carbohydrate level suggests that a carnivorous fish like Sparus aurata can tolerate partial replacement of protein by carbohydrate in the commercial diets supplied in culture. The relationship between enzyme activities, ration size and fish growth indicates that the enzymes quickly respond to dietary manipulations of cultured fish.

Distinctive response of naïve lymphocytes from cord blood to primary activation via TCR

Umbilical cord blood (UCB) is now being considered an alternative to bone marrow for restoring hematopoiesis after myeloablative therapy. The lower risk of acute and chronic graft‐versus‐host disease in patients who received UCB cells seems related to the nature of UCB–T cells. Phenotypically, UCB–CD3+ cells are mostly naive (CD45RA+) and represent a transitional population between thymocytes and adult T cells. We examined the immune reactivity of highly purified, negatively selected CD4+CD45RA+ cells by mimicking activation via T cell receptor (TCR). All experiments included the extensively characterized adult peripheral blood (APB) cells as reference. On the contrary to APB, naive UCB–CD4+ cells were able to proliferate with anti‐CD3 stimulation alone. With addition of interleukin (IL)‐2 or costimulatory signal, both populations reached similar proliferation. Forty‐eight hours after anti‐CD3 stimulation, CD4+CD45RA+ from UCB, but not APB, showed characteristic blastic morphology and significant expression of CD25 on the surface. A low concentration of IL‐2 was detected at 24 h by anti‐CD3‐stimulated UCB CD4+CD45RA+, which rapidly disappeared. By 72 h after activation, CD4+CD45RA+ UCB cells showed extensive apoptosis, whereas CD4+CD45RA+ APB cells showed low levels of apoptosis. Using RNase protection assay, we observed that CD95L levels were significantly higher in naive CD4+ cells from UCB than from APB after activation. However, neutralizing Fas‐Fc protein was unable to inhibit anti‐CD3‐induced apoptosis, suggesting that this was a CD95‐independent mechanism. These results indicate that UCB–CD4+CD45RA+ cells are able to start proliferating as a result of early IL‐2 production after TCR engagement alone, but probably, as a result of the consumption of this IL‐2, they undergo cell death.

Functional consequences of CD36 downregulation by TLR signals

TLR recognition activates the secretion of pro- and anti-inflammatory cytokines and it also modulates the expression of crucial molecules involved in phagocytosis and antimicrobial activity. Scavenger receptors can act as TLR co-receptors or facilitate antigen loading. However, it remains unknown whether TLR can modulate the expression of these scavenger receptors. We stimulated human peripheral blood mononuclear cells (PBMC) with TLR2 (Pam3CSK4 and FSL1) and TLR4 ligand lipopolysaccharide (LPS) and then analyzed CD36 expression on different monocyte subpopulations by flow cytometry. TLR2 and TLR4 ligands can downregulate CD36 on the surface of monocytes, guiding the protein to intracellular compartments. Even though TLR-activation induced TNFα, IL-10 and IL-6 production, only recombinant TNFα was able to downregulate CD36. Neutralizing anti-TNFα antibodies showed that the Pam3CSK4 and FSL1-induced downregulation was partially mediated by TNFα but not by IL-6 or IL-10. However, LPS-induced downregulation could have also been caused by direct TLR4 targeting and signaling, and/or mediated by other unknown factors. CD36 downregulation reduced the capability of monocytes to phagocyte apoptotic neutrophils. In conclusion, modulation of scavenger receptor expression by TLR targeting on monocytes has functional consequences. Characterization this complex regulation may help us to understand this innate response and develop specific therapeutic drugs for each mechanism.

MDP-induced Selective Tolerance to TLR4 Ligands: Impairment in NOD2 Mutant Crohn's Disease Patients

Background: Pathogen infection is a complex process in which several pathogen‐recognition receptor (PRR) pathways are activated to induce proinflammatory mediators. The activation of multiple PRRs suggests an interaction between Toll‐like receptors (TLRs) and nucleotide‐binding oligomerization domain‐like receptor (NOD) signaling pathways. Methods: To understand the modulation induced by NOD2 signals on successive responses to pathogen‐associated molecular patterns (PAMPs), we examined how muramyl dipeptide (MDP) pretreatment reprograms the MDP+LPS (lipopolysaccharide) response of monocytes from human peripheral blood. Results: Preexposure to bacterial MDP components induced selective tolerance to a subsequent NOD2+TLR4 stimulation. MDP pretreatment inhibited the production of tumor necrosis factor alpha (TNF&agr;) and interleuken 10 (IL10), whereas IL6 and IL8 remained unaffected. MDP‐induced tolerance was independent of receptor downregulation but was associated with reduced levels of phosphorylated TAK1 and abrogated phosphorylation of the downstream MAPK. Since Nod2 mutations have been associated with susceptibility to develop Crohns disease (CD), we compared the MDP‐induced tolerance in healthy donors and CD patients with compound heterozygous Nod2 mutations (Mut‐Nod2) expressing variant NOD2 proteins. MDP‐induced tolerance in Mut‐Nod2 patients reduced IL10 but not TNF&agr; production. In contrast with healthy donors, a p38‐independent TNF&agr; production was observed during the kinetics of the MDP+LPS response in Mut‐Nod2 patients. Conclusions: Our findings suggest that the selective tolerance induced by MDP in healthy donors was related to the modulation of a convergent nub of NOD2 and TLR4 signaling pathways. This MDP‐induced tolerance was impaired in Mut‐Nod2 CD patients, resulting in a p38‐independent TNF&agr; production and an imbalance between pro‐ and antiinflammatory cytokines that could be partly responsible for the pathogenesis of CD. Inflamm Bowel Dis 2009

Functional consequences of platelet binding to T lymphocytes in inflammation

Expression of the scavenger receptor CD36 on lymphocytes is intriguing. We observed that a minor subpopulation of lymphocytes expressed CD36 on the cell surface. We investigated the source of CD36 and also the proliferation and cytokine production of these CD36+ CD4+ lymphocytes. Flow cytometry analysis and immunofluorescence microscopy showed that CD36+ platelets were responsible for CD36 detection on lymphocytes. CD36 was then used as a tool to characterize lymphocytes with bound platelets. Activation‐induced proliferation was lower in CD4+ lymphocytes with bound platelets than lymphocytes without bound platelets. IL‐17 and IFN‐γ production was also reduced in lymphocytes with bound platelets. We then studied the presence of CD36+ CD4+ lymphocytes in RA patients. We observed that the percentage of CD4+ lymphocytes with bound platelets was higher on RA patients than in healthy donors. RA patients with higher titers of anti‐CCP, RF levels, and cardiovascular risk index presented a lower percentage of CD4+ lymphocytes with bound platelets. These patients also had higher IL‐17 and IFN‐γ production. These results suggest that platelet‐binding modifies lymphocyte function. This binding could be a regulatory mechanism in RA that confers a less severe phenotype.

IL-6 blockade reverses the abnormal STAT activation of peripheral blood leukocytes from rheumatoid arthritis patients.

Considering the interplay of multiple STATs in response to cytokines, we investigated how IL-6 and its blocking affect STAT signaling in rheumatoid arthritis (RA). Leukocytes obtained from RA patients before and after tocilizumab treatment and healthy donors (HDs) were cytokine-stimulated and STAT phosphorylation was analyzed by cytometry. RA patients had significantly fewer pSTAT1+, pSTAT3+, and pSTAT6+ monocytes and pSTAT5+ lymphocytes than HDs. After 24weeks of treatment, percentages of IFNγ-induced pSTAT1+ and IL-10-induced pSTAT3+ monocytes in RA patients increased, reaching levels comparable to HDs. pSTAT1+ and pSTAT3+ cells correlated inversely with RA disease activity index and levels of pSTAT+ cells at baseline were higher in patients with good EULAR response to tocilizumab. IFNγ-induced pSTAT1+ cells correlated inversely with memory T cells and anti-CCP levels. IL-10-induced pSTAT3+ cells correlated with Treg/Teff ratio. Our findings suggest that IL-6 blocking reduces the inflammatory mechanisms through the correction of STAT1 and STAT3 activation status.

Interleukin-19 Impairment in Active Crohn’s Disease Patients

The exact function of interleukin-19 (IL-19) on immune response is poorly understood. In mice, IL-19 up-regulates TNFα and IL-6 expression and its deficiency increases susceptibility to DSS-induced colitis. In humans, IL-19 favors a Th2 response and is elevated in several diseases. We here investigate the expression and effects of IL-19 on cells from active Crohn’s disease (CD) patient. Twenty-three active CD patients and 20 healthy controls (HC) were included. mRNA and protein IL-19 levels were analyzed in monocytes. IL-19 effects were determined in vitro on the T cell phenotype and in the production of cytokines by immune cells. We observed that unstimulated and TLR-activated monocytes expressed significantly lower IL-19 mRNA in active CD patients than in HC (logFC = −1.97 unstimulated; −1.88 with Pam3CSK4; and −1.91 with FSL-1; p<0.001). These results were confirmed at protein level. Exogenous IL-19 had an anti-inflammatory effect on HC but not on CD patients. IL-19 decreased TNFα production in PBMC (850.7±75.29 pg/ml vs 2626.0±350 pg/ml; p<0.01) and increased CTLA4 expression (22.04±1.55% vs 13.98±2.05%; p<0.05) and IL-4 production (32.5±8.9 pg/ml vs 13.5±2.9 pg/ml; p<0.05) in T cells from HC. IL-10 regulated IL-19 production in both active CD patients and HC. We observed that three of the miRNAs that can modulate IL-19 mRNA expression, were up-regulated in monocytes from active CD patients. These results suggested that IL-19 had an anti-inflammatory role in this study. Defects in IL-19 expression and the lack of response to this cytokine could contribute to inflammatory mechanisms in active CD patients.

Rituximab-induced interleukin-15 reduction associated with clinical improvement in rheumatoid arthritis

Rituximab therapy alters all aspects of B‐cell participation in the disturbed immune response of rheumatoid arthritis patients. To determine the impact of B‐cell depletion on other immune compartments, we analysed levels of soluble and surface interleukin‐15 (IL‐15) along with the frequency of IL‐15‐related subsets after rituximab treatment. We then studied the correlation of observed changes with clinical activity. Heparinized blood samples from 33 rheumatoid arthritis patients were collected on days 0, 30, 90 and 180 after each of three rituximab cycles. Serum cytokine levels were determined by ELISA. Interleukin‐15 trans‐presentation was analysed by cytometry. Flow cytometry with monoclonal antibodies was performed to analyse circulating cell subsets. Interleukin‐15 was detected in the serum of 25 patients before initiating the treatment. Rituximab then progressively reduced serum IL‐15 (138 ± 21 pg/ml at baseline, 48 ± 18 pg/ml after third cycle, P = 0·03) along with IL‐17 (1197 ± 203 pg/ml at baseline, 623 ± 213 pg/ml after third cycle, P = 0·03) and tended to increase the frequency of circulating regulatory T cells (3·1 ± 1 cells/μl at baseline, 7·7 ± 2 cells/μl after third cycle). Rituximab also significantly decreased IL‐15 trans‐presentation on surface monocytes of patients negative for IL‐15 serum (mean fluorescence intensity: 4·82 ± 1·30 at baseline, 1·42 ± 0·69 after third cycle P = 0·05). Reduction of serum IL‐15 was associated with decrease in CD8+ CD45RO+/RA+ ratio (1·17 ± 0·21 at baseline, 0·36 ± 0·06 at third cycle, P = 0·02). DAS28, erythrocyte sedimentation rate and C‐reactive protein correlated significantly with CD8+ CD45RO+/RA+ ratio (R = 0·323, R = 0·357, R = 0·369 respectively, P < 0·001). Our results suggest that sustained clinical improvement after rituximab treatment is associated with IL‐15/memory T‐cell‐related mechanisms beyond circulating B cells.

Binding of Platelets to Lymphocytes: A Potential Anti-Inflammatory Therapy in Rheumatoid Arthritis

Soluble factors released from platelets can modulate the immune response of leukocytes. We and others have recently found that T lymphocytes with bound platelets have reduced proliferation and IFN-γ and IL-17 production. Thus, we speculate that if we induce the binding of platelets to lymphocytes, we will be able to regulate the inflammatory response. When we cocultured platelets with lymphocytes at different ratios, we were able to increase the percentage of lymphocytes with bound platelets. The coculture of platelets with lymphocytes in the presence of stimulation decreased the production of IFN-γ and TNF-α, T cell proliferation, and the expression of CD25, PD-L1, and SLAM. However, this coculture increased CD39 expression. All of these effects were dependent on the dose of platelets and operated indistinctly with platelets from different healthy donors. When platelets were cocultured in the same compartment with lymphocytes, we observed less IFN-γ and TNF-α production and T lymphocyte proliferation than in cultures with platelets separated from lymphocytes by a 0.4-μm pore size filter. The binding of platelets to lymphocytes was blocked with anti–P-selectin Abs, and when this occurred we observed higher IFN-γ and TNF-α production than in nonblocked conditions. The cocultures of platelets with synovial fluid cells from rheumatoid arthritis patients reduced inflammatory cytokine production and increased IL-10 production. These results suggest that platelet binding to lymphocytes effectively regulates T lymphocyte function. This mechanism could be easily applied to reduce inflammatory responses.

Adalimumab regulates intracellular TNFα production in patients with rheumatoid arthritis.

IntroductionAdalimumab is a fully human anti–tumor necrosis factor α (anti-TNFα) monoclonal antibody that specifically blocks the interaction of TNFα with its receptors. It binds both soluble and transmembrane TNFα. We hypothesized that blocking these TNFα signals regulates the altered TNFα production in rheumatoid arthritis (RA) patients.MethodsWe compared, by flow cytometry, Toll-like receptor induction levels of membrane and intracellular TNFα in monocytes (iTNFα + CD14+ cells) from 12 patients before and after adalimumab treatment with those from 5 healthy donors.ResultsBefore starting the treatment, the percentage of iTNFα+ CD14+ cells in the RA patients was significantly lower than that in healthy donors (mean ± SEM = 33.16 ± 4.82% vs 66.51 ± 2.4%, P < 0.001). When we added in vitro TNFα to healthy donor culture cells, levels of iTNFα+ CD14+ cells decreased, suggesting that the TNFα signal was responsible for the iTNFα+ CD14+ cell downregulation observed in the RA patients. After 2, 6 and 12 adalimumab injections, we observed significant blocking of membrane and soluble TNFα and a progressive increase in iTNFα+ CD14+ cells in ten patients with a good to moderate response as defined by the European League Against Rheumatism (EULAR) criteria. Levels of iTNFα+ CD14+ cells after 12 injections in these 10 patients were comparable to levels in healthy donors. In two patients, iTNFα+ CD14+ cell upregulation was not observed, and their EULAR-defined responses had not improved. The first patient developed antiadalimumab antibodies, explaining why adalimumab was not able to block membrane and soluble TNFα. In the second patient, adalimumab was discontinued because of adverse effects, which led to a decrease in iTNFα+ CD14+ cells to levels measured before treatment.ConclusionsOur findings suggest that adalimumab treatment in RA patients can return iTNFα levels to those of healthy donors. This effect was not observed in the presence of neutralizing antiadalimumab antibodies.