Biomedical subjects
Publications and source records attributed to Juan Rivera.
We report the case of a 29-year-old man who developed acute congestive heart failure secondary to cardiac Chagas' disease in the setting of Trypanosoma cruzi reactivation by acquired immune deficiency syndrome.
The high affinity IgE Fc receptor (FcepsilonRI) beta chain functions as a signal amplifier and has been linked to atopy, asthma, and allergy. Herein, we report on a previously unrecognized negative regulatory role for the nonconventional beta chain immunoreceptor tyrosine-based activation motif that contains three tyrosine residues (YX5YX3Y). Degranulation and leukotriene production was found to be impaired in cells expressing the mutated FcepsilonRIbeta immunoreceptor tyrosine-based activation motifs FYY, YYF, FYF, and FFF. In contrast, cytokine synthesis and secretion were enhanced in the YFY and FFF mutants. FcepsilonRI phosphorylation and Lyn kinase co-immunoprecipitation was intact in the YFY mutant but was lost in the FYF and FFF mutants. The phosphorylation of Syk, LAT, phospholipase gamma1/2, and Srchomology 2 domain-containing protein phosphatase 2 was intact, whereas the phosphorylation of SHIP-1 was significantly reduced in the YFY mutant cells. The FYF and FFF mutants were defective in phosphorylating all of these molecules. In contrast, the phosphorylation of ERK, p38 MAPK, IkappaB kinase beta (IKKbeta), and nuclear NFkappaB activity was enhanced in the YFY and FFF mutants. These findings show that the FcepsilonRIbeta functions to both selectively amplify (degranulation and leukotriene secretion) and dampen (lymphokine) mast cell effector responses.
A role for Lyn kinase as a positive regulator of immunoglobulin (Ig)E-dependent allergy has long been accepted. Contrary to this belief, Lyn kinase was found to have an important role as a negative regulator of the allergic response. This became apparent from the hyperresponsive degranulation of lyn-/- bone marrow-derived mast cells, which is driven by hyperactivation of Fyn kinase that occurs, in part, through the loss of negative regulation by COOH-terminal Src kinase (Csk) and the adaptor, Csk-binding protein. This phenotype is recapitulated in vivo as young lyn-/- mice showed an enhanced anaphylactic response. In vivo studies also demonstrated that as lyn-/- mice aged, their serum IgE increased as well as occupancy of the high affinity IgE receptor (FcepsilonRI). This was mirrored by increased circulating histamine, increased mast cell numbers, increased cell surface expression of the high affinity IgE receptor (FcepsilonRI), and eosinophilia. The increased IgE production was not a consequence of increased Fyn kinase activity in lyn-/- mice because both lyn-/- and lyn-/- fyn-/- mice showed high IgE levels. Thus, lyn-/- mice and mast cells thereof show multiple allergy-associated traits, causing reconsideration of the possible efficacy in therapeutic targeting of Lyn in allergic disease.
Mast cells secrete various substances that initiate and perpetuate allergic responses. Cross-linking of the high-affinity receptor for IgE (FcepsilonRI) in RBL-2H3 and bone marrow-derived mast cells activates sphingosine kinase (SphK), which leads to generation and secretion of the potent sphingolipid mediator, sphingosine-1-phosphate (S1P). In turn, S1P activates its receptors S1P1 and S1P2 that are present in mast cells. Moreover, inhibition of SphK blocks FcepsilonRI-mediated internalization of these receptors and markedly reduces degranulation and chemotaxis. Although transactivation of S1P1 and Gi signaling are important for cytoskeletal rearrangements and migration of mast cells toward antigen, they are dispensable for FcepsilonRI-triggered degranulation. However, S1P2, whose expression is up-regulated by FcepsilonRI cross-linking, was required for degranulation and inhibited migration toward antigen. Together, our results suggest that activation of SphKs and consequently S1PRs by FcepsilonRI triggering plays a crucial role in mast cell functions and might be involved in the movement of mast cells to sites of inflammation.
FcepsilonRI expression and function is a central aspect of allergic disease. Using bone marrow-derived mouse mast cell populations, we have previously shown that the Th2 cytokine IL-4 inhibits FcepsilonRI expression and function. In the current study we show that the Th2 cytokine IL-10 has similar regulatory properties, and that it augments the inhibitory effects of IL-4. FcepsilonRI down-regulation was functionally significant, as it diminished inflammatory cytokine production and IgE-mediated FcepsilonRI up-regulation. IL-10 and IL-4 reduced FcepsilonRI beta protein expression without altering the alpha or gamma subunits. The ability of IL-4 and IL-10 to alter FcepsilonRI expression by targeting the beta-chain, a critical receptor subunit known to modulate receptor expression and signaling, suggests the presence of a Th2 cytokine-mediated homeostatic network that could serve to both initiate and limit mast cell effector function.
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BACKGROUND: Iron supplements improve hemoglobin status and reduce anemia due to iron deficiency. It is not known whether multiple micronutrient (MM) supplements are as efficacious as are iron supplements alone in improving hemoglobin concentrations. OBJECTIVE: We conducted a randomized, double-blind community trial in Mexico to compare the efficacy of MM supplements containing iron with that of iron alone in improving hemoglobin concentrations in nonpregnant women. DESIGN: Nonpregnant women (n = 158) were recruited from a semirural community in Mexico and were randomly assigned to receive iron alone (60 mg; Fe group) or MM supplements (vitamins A, B complex, C, D, E, and K and iron, zinc, and magnesium; MM group) 6 d/wk in their home for 12 wk. Hemoglobin concentrations were measured in capillary blood samples at baseline and follow-up. RESULTS: The treatment groups (MM: n = 75; Fe: n = 77) did not differ significantly at recruitment in age, schooling, literacy, or socioeconomic status. There were no significant differences between groups in compliance (median: 97.5%), baseline hemoglobin concentrations, or prevalence of anemia (20%). Losses to follow-up (4%) and mean (+/-SD) changes in hemoglobin (MM group: 6.7 +/- 10.6 g/L; Fe group: 7.1 +/- 13.6 g/L) were not significantly different between groups. However, the change in hemoglobin in anemic subjects was greater in the Fe group than in the MM group (P < 0.05 for interaction), and there was no significant difference in nonanemic subjects. CONCLUSIONS: MM supplements may not be as efficacious as is iron alone in improving the hemoglobin status of anemic women.
The impact of iron-only supplements (FE) versus multiple micronutrient supplements containing iron (MM) during pregnancy on iron status was assessed in a subsample (n = 453) of women who participated in a randomized double-blind trial in Mexico. Compliance, monitored by observation, was high (>85%). The two groups were similar at recruitment (<13 wk gestation) for various sociodemographic characteristics and for mean hemoglobin (Hb) concentrations and prevalence of anemia (Hb < 110 g/L; 11%). However, mean serum ferritin was higher (P < 0.05) in the MM group (n = 142) compared to the FE group (n = 148) and the prevalence of iron deficiency (serum ferritin < 12 micro g/L) was lower in the MM group (44.4%) compared to the FE group (57.4%). By the third trimester, almost half the women were anemic in both groups, and mean Hb (g/L) was lower for the MM group (104.2; 95% CI: 102.5, 106.0) compared to the FE group (108.1; 95% CI: 106.4, 109.8) after adjusting for baseline serum ferritin. In contrast, there were no differences in Hb concentrations at 1 mo postpartum or in mean ferritin and prevalence of iron deficiency at 32 wk gestation and 1 mo postpartum (90.9 and 45.1% for the MM group; 92.6 and 47.3% for the FE group, respectively). In conclusion, rather than improve Hb or iron status relative to FE-only supplements as hypothesized, MM supplements may have slightly reduced Hb concentrations during pregnancy. Neither supplement was able to meet iron needs as evidenced by dramatic increases in anemia and iron deficiency by the end of pregnancy.
Sphingosine kinase has been recognized as an essential signaling molecule that mediates the intracellular conversion of sphingosine to sphingosine-1-phosphate. In mast cells, induction of sphingosine kinase and generation of sphingosine-1-phosphate have been linked to the initial rise in Ca(2+), released from internal stores, and to degranulation. These events either precede or are concomitant with the activation of phospholipase C-gamma and the generation of inositol trisphosphate. Here we show that sphingosine kinase type 1 (SPHK1) interacts directly with the tyrosine kinase Lyn and that this interaction leads to the recruitment of this lipid kinase to the high-affinity receptor for immunoglobulin E (FcepsilonRI). The interaction of SPHK1 with Lyn caused enhanced lipid and tyrosine kinase activity. After FcepsilonRI triggering, enhanced sphingosine kinase activity was associated with FcepsilonRI in sphingolipid-enriched rafts of mast cells. Bone marrow-derived mast cells from Lyn(-/)(-) mice, compared to syngeneic wild-type cells, were defective in the initial induction of SPHK1 activity, and the defect was overcome by retroviral Lyn expression. These findings position the activation of SPHK1 as an FcepsilonRI proximal event.
Antigen-induced cross-linking of immunoglobulin E (IgE) antibodies bound to the high-affinity IgE receptor (FcepsilonRI), on mast cells results in the release of mediators that initiate an inflammatory response. This normal immune response has been abducted by immunological adaptation, through the production of IgE antibodies to normally innocuous substances, to cause allergic disease. Therefore, understanding the molecular requirements in IgE-dependent mast-cell activation holds promise for therapeutic intervention in disease. Recent investigation on the functional coupling of FcepsilonRI to the intracellular signaling apparatus has provided paradigm-altering insights on the importance and function of Src family protein tyrosine kinases (Src PTK) in mast-cell activation. In this synopsis, we review the current knowledge on the role of the Src PTKs, Fyn and Lyn, in mast-cell activation and discuss the implications of our findings on allergic disease.
Mast cells are central to allergic disease. Their immediate (exocytosis of granule-stored allergic-mediators) and delayed (de novo synthesis of inflammatory mediators) response to an allergen underlies the symptoms seen in acute; and chronic allergic disease. Thus, intervention in the allergen-mediated activation of mast cells is a long sought after goal in the treatment and management of allergic disease. The recent gain in deciphering the molecular mechanisms underlying immunoglobulin E (IgE)-mediated mast cell activation has provided optimism for the development of new therapeutic strategies. Among the most promising is the use of humanized anti-IgE antibodies that inhibit binding of IgE to its high affinity receptor (FcepsilonRI) on the mast cell. Other strategies target molecules proximal to FcepsilonRI, whose activities are central in mast cell activation. One such molecule, Syk kinase, has been targeted by various approaches including a small molecule inhibitor that specifically abrogates mast cell degranulation. More recently, various molecules that function to promote protein-protein interactions (adapters) were demonstrated as essential to mast cell degranulation and cytokine production. It remains to be seen if these molecules hold therapeutic promise for disease intervention. Additional studies identifying molecules required for mast cell granule fusion and content exocytosis also bodes well for discovery of new therapeutic targets. While our understanding of IgE-mediated mast cell activation is still at its inception, the modest success in identifying molecules essential to this process affords some confidence for better treatment of allergic disease.
The linker for activation of T cells (LAT) is an adaptor protein critical for Fc epsilon RI-mediated mast cell activation. LAT is a substrate of the tyrosine kinases activated after TCR and Fc epsilon RI engagement. After phosphorylation of the cytosolic domain of LAT, multiple signaling molecules such as phospholipase C-gamma1, Grb2, and Gads associate with phosphorylated LAT via their SH2 domains. The essential role of the four distal tyrosines in TCR-mediated signaling and T cell development has been demonstrated by experiments using LAT-deficient cell lines and genetically modified mice. To investigate the role of these four tyrosines of LAT in Fc epsilon RI-mediated mast cell activation, bone marrow-derived mast cells from LAT-deficient mice were infected with retroviral vectors designed to express wild-type or mutant LAT. Examination of bone marrow-derived mast cells expressing various tyrosine to phenylalanine mutants in LAT demonstrates a differential requirement for these different binding sites. In these studies, assays of biochemical pathways, degranulation, and cytokine and chemokine release were performed. Finally, the role of these tyrosines was also evaluated in vivo using genetically modified animals. Deletion of all four distal tyrosines, and in particular, loss of the primary phospholipase C-gamma-binding tyrosine had a significant effect on antigen-induced histamine release.
Exocytosis of mast cell granules requires a vesicular- and plasma membrane-associated fusion machinery. We examined the distribution of SNARE membrane fusion and Munc18 accessory proteins in lipid rafts of RBL mast cells. SNAREs were found either excluded (syntaxin2), equally distributed between raft and non-raft fractions (syntaxin4, VAMP-8, VAMP-2), or selectively enriched in rafts (syntaxin3, SNAP-23). Syntaxin4-binding Munc18-3 was absent, whereas small amounts of the syntaxin3-interacting partner Munc18-2 consistently distributed into rafts. Cognate SNARE complexes of syntaxin3 with SNAP-23 and VAMP-8 were enriched in rafts, whereas Munc18-2/syntaxin3 complexes were excluded. This demonstrates a spatial separation between these two types of complexes and suggests that Munc18-2 acts in a step different from SNARE complex formation and fusion.
Mast cell degranulation and de novo cytokine production is a consequence of antigen-aggregation of the immunoglobulin E (IgE)-occupied high affinity receptor for IgE (Fc epsilon RI). Herein, we report that lymphokines that promote allergic inflammation, like MCP-1, were potently induced at low antigen (Ag) concentrations or at low receptor occupancy with IgE whereas some that down-regulate this response, like interleukin (IL)-10, required high receptor occupancy. Weak stimulation of mast cells caused minimal degranulation whereas a half-maximal secretory response was observed for chemokines and, with the exception of TNF-alpha, a weaker cytokine secretory response was observed. The medium from weakly stimulated mast cells elicited a monocyte/macrophage chemotactic response similar to that observed at high receptor occupancy. Weak stimulation also favored the phosphorylation of Gab2 and p38MAPK, while LAT and ERK2 phosphorylation was induced by a stronger stimulus. Gab2-deficient mast cells were severely impaired in chemokine mRNA induction whereas LAT-deficient mast cells showed a more pronounced defect in cytokines. These findings demonstrate that perturbation of small numbers of IgE receptors on mast cells favors certain signals that contribute to a lymphokine response that can mediate allergic inflammation.
Compound exocytosis of inflammatory mediators from mast cells requires SNARE and a series of accessory proteins. However, the molecular steps that regulate secretory granule movement and membrane fusion as well as the role of the cytoskeleton are still poorly understood. Here, we report on our investigation of the role of syntaxin-binding Munc18 isoforms and the microtubule network in this process. We found that mast cells express Munc18-2, which interacts with target SNAREs syntaxin 2 or 3, as well as Munc18-3, which interacts with syntaxin 4. Munc18-2 was localised to secretory granules, whereas Munc18-3 was found on the plasma membrane. Increased expression of Munc18-2 and derived peptides containing an interfering effector loop inhibited IgE-triggered exocytosis, while increased expression of Munc18-3 showed no effect. Munc18-2 localisation on granules is polarised; however, upon stimulation Munc18-2 redistributed into forming lamellipodia and persisted on granules that were aligned along microtubules, but was excluded from F-actin ruffles. Disruption of the microtubule network with nocodazole provoked Munc18-2 redistribution and affected mediator release. These findings suggest a role for Munc18-2 and the microtubule network in the regulation of secretory granule dynamics in mast cells.
Mast cells are exposed to an oxidative environment in the course of allergic and inflammatory reactions. We have examined the effects of H(2)O(2) stimulation in a primary rat basophilic leukemia cell line (RBL-2H3) and compared with IgE-dependent stimulation. Like IgE stimulation, H(2)O(2) up-regulates IL-4 and IL-6 gene expression and cytokine secretion, shows a little effect on IL-5 but does not induce IL-10 gene expression. Simultaneous H(2)O(2) treatment and FcepsilonRI triggering of mast cells has additive effects on IL-4 expression. In addition, we show that both stimuli induce the nuclear translocation of APE/Ref-1, a bifunctional enzyme that stimulates the DNA-binding activity of several transcription factors through the reduction of highly reactive cysteines. Conditional inactivation of APE/Ref-1 expression abolishes H(2)O(2)-induced IL-4 and IL-6 gene expression but does not affect that induced by FcepsilonRI stimulation. Our findings indicate that oxidative stress activates the gene expression of a specific cytokine pattern in mast cells through an APE/Ref-1-dependent pathway, which is distinct from the one that is activated by FcepsilonRI stimulation. Nonetheless, H(2)O(2) and FcepsilonRI signalings are additive in augmenting IL-4 production. Most importantly, oxidative stress can induce a pro-type 2 inflammatory response from mast cells that is independent of FcepsilonRI stimulation.
BACKGROUND: Tissue iron depletion may negatively affect endurance performance and muscle fatigability. OBJECTIVE: We investigated tissue-level iron depletion and progressive fatigue of the quadriceps during dynamic knee-extension exercise in young women. DESIGN: Twenty iron-depleted (serum ferritin < 20 micro g/L), nonanemic (hemoglobin > 110 g/L) women (macro x +/- SEM age: 29.1 +/- 1.2 y) received iron (iron group) or placebo (placebo group) for 6 wk in a randomized, double-blind trial (n = 10 per group). A protocol integrating 2-3-s maximal voluntary static contractions (MVCs) with dynamic knee extensions was used to assess fatigue. RESULTS: No significant differences between the groups in baseline iron status, MVC at rest, or MVC at the end of the protocol were observed. After treatment, serum iron and transferrin saturation increased significantly in the iron group (P = 0.02 and P = 0.03, respectively). Serum transferrin receptor concentrations increased significantly in the placebo group (P < 0.01) but not in the iron group. After treatment, the rate of decrease in MVC was attenuated in the iron group but not in the placebo group (P = 0.01). In the iron group, MVC at the sixth minute of the fatigue protocol and MVC at the end of the protocol were approximately 15% (P = 0.04) and approximately 27% higher (P < 0.01), respectively, after treatment. These improvements were not related to changes in iron-status indexes or tissue iron stores, although power was low (< 0.50) to detect these relations. CONCLUSIONS: Iron supplementation was associated with a significant improvement in muscle fatigability. Interpretation regarding the direct role of tissue iron status is limited by the study's low power to detect relations between tissue iron improvement and decreased muscle fatigue.
BACKGROUND: Little is known about the benefits of prenatal multivitamin and mineral supplements in reducing low birth weight. OBJECTIVE: We conducted a randomized, double-blind clinical trial in semirural Mexico to compare the effects of multiple micronutrient (MM) supplements with those of iron supplements during pregnancy on birth size. DESIGN: Pregnant women (n = 873) were recruited before 13 wk of gestation and received supplements 6 d/wk at home, as well as routine antenatal care, until delivery. Both supplements contained 60 mg Fe, but the MM group also received 1-1.5 times the recommended dietary allowances of several micronutrients. RESULTS: At recruitment, the women in the 2 groups were not significantly different in age, parity, economic status, height, or hemoglobin concentration but differed significantly in marital status (4.6% and 2.0% of women in the MM and iron-only groups, respectively, were single mothers) and mean (+/- SD) body mass index (in kg/m(2); 24.6 +/- 4.3 and 23.8 +/- 3.9 in the iron-only and MM groups, respectively). Losses to follow-up (25%) and compliance (95%) did not differ significantly between the groups. In intent-to-treat analyses (MM group: n = 323; iron-only group: n = 322), mean (+/- SD) birth weight (2.981 +/- 0.391 and 2.977 +/- 0.393 kg in the MM and iron-only groups, respectively) and birth length (48.61 +/- 1.82 and 48.66 +/- 1.83 cm in the MM and iron-only groups, respectively) did not differ significantly between the groups. CONCLUSION: These findings suggest that MM supplementation during pregnancy does not lead to greater infant birth size than does iron-only supplementation.