Respuesta inmune respiratoria al año de vida en el niño prematuro / Respiratory immune response in first year of a preterm infant

Introducción: Existe un gran desconocimiento acerca de la evolución del sistema inmune en la mucosa respiratoria del niño prematuro a largo plazo. La inmadurez y las infecciones respiratorias pueden influir sobre la respuesta inmune de las mucosas. El propósito de este estudio era evaluar la secreción respiratoria de los mediadores inmunológicos al año de vida en niños prematuros. Pacientes y métodos: Desde octubre de 2008 hasta abril de2009 se reclutaron 77 prematuros nacidos en 6 servicios de pediatría de Castilla y León, así como 14 controles sanos a término. Los prematuros fueron citados al año de edad gestacional corregida y los niños a término al año de vida, momento en el cual se les realizó un lavado nasal para determinar los niveles de 27 mediadores inmunológicos mediante un ensayo de Biorad®. Resultados: Los niños prematuros tenían niveles más elevados de quimiocinas (eotaxina, IP-10), citocinas Th-1 (IFN-epsilon), Th-2 (IL-13), Th-17 (IL-17) y factores de crecimiento celular (PDGF-bb, VEGF, FGF-b, G-CSF y GM-CSF) que los niños a término. Cuando se compararon los niveles de mediadores entre los niños que habían recibido profilaxis para el virus respiratorios incitial con palivizumab y los que no, los segundos tenían niveles significativamente más altos de MCP-1, IL-1RA, IL-10,IL-12p70 y VEGF (p <0,05) que los primeros. Conclusiones: Este trabajo demuestra por vez primera la influencia de la prematuridad sobre los perfiles de secreción respiratoria de las citocinas y quimiocinas a largo plazo. Por otra parte, nuestros resultados indican que la evaluación del impacto de la profilaxis de la infección respiratoria es un camino interesante para comprender la maduración de la respuesta inmune de la mucosa respiratoria del prematuro(AU)

Introduction: There is a big unawareness about a long term respiratory mucous immune system evolution in the preterm infant. Immaturity and respiratory infections can have a big influence on the mucous immune responses. This investigation’s purpose is the evaluation of respiratory secretion of inflammatory immunological mediators in the first year of a preterm infant. Patients and methods: Between October 2008 and April 2009, 77 preterm infants were born in 6 pediatric services of Castilla y Leon, plus to another 14 healthy controls results. Children were invited on their first corrected gestational age and the ones of healthy controls results. Nasal washing were applied to determine 27 immunological mediators’ levels by applying a Biorad test. Results: The preterm infants has higher chemokine (eotaxin,IP-10), cytokines Th-1 (IFN-epsilon), Th-2 (IL-13), Th-17 (IL-17) and cell growing factors (PDGF-bb, VEGF, FGF-b, G-CSF and GM-CSF)levels than a healthy control results children. When a comparison was made between children that received prophylaxis for their respiratory syncytial virus with palivizumab and the ones that did not receive it, the second group showed higher MCP-1, IL-1RA, IL-10, IL-12p70 and VEGF (p <0,05) levels. Conclusions: This work proves, for the first time, the influence of the premature birth on chemokine and cytokines respiratory secretion levels in a long term concepts. On other hand, our results indicate that prophylaxis impact in the respiratory infection is an interesting way to understand respiratory mucous immune response maturation in the preterm infant(AU)

The NRF2 gene variant, -653G/A, is associated with nephritis in childhood-onset systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease associated with oxidative stress and characterized by chronic inflammation. Kidney malfunction, an aggressive characteristic of this disease, is not present in all affected individuals. The Nrf2-Keap1 pathway is important in protecting against oxidative stress and inflammation. Mouse models and genome-wide scans have suggested NRF2 (Nuclear factor (erythroid-derived 2)-like 2) as a candidate gene for susceptibility to SLE. We therefore investigated whether NRF2 polymorphisms are associated with childhood-onset SLE in a Mexican Mestizo population. Two single nucleotide polymorphisms (SNPs) were genotyped by TaqMan((R)) assays in 362 patients with childhood-onset SLE and 379 controls. We found no significant association between susceptibility to SLE and NRF2 polymorphisms. However, after population stratification by gender, the heterozygous genotype of the -653G/A SNP was significantly associated with nephritis in females only [OR = 1.81, CI (1.04-3.12), p = 0.032]. This association was stronger in females affected with severe nephritis [classes IV-VI; OR = 2.16, CI (1.12-4.15), p = 0.019]. Our results suggest that NRF2 is not associated with susceptibility to childhood-onset SLE, but it could confer a risk for developing kidney malfunction in SLE-affected individuals.

Diagnóstico de rabdomioma fetal ecográfico / Ultrasonographic diagnosis of fetal rhabdomyoma

El rabdomioma es una tumoración rara detectada, en la mayor parte de las ocasiones, por ecografía y representa aproximadamente un 1% del total de las anomalías cardíacas halladas intraútero. La evolución intrauterina y su repercusión hemodinámica dependen del crecimiento del tumor. Grandes masas pueden ser causa de obstrucciones y posteriormente de hidropesía y taquiarritmias. A estas complicaciones se añade su asociación a la esclerosis tuberosa, la cual ocupa un lugar importante en el consejo prenatal a los padres, ya que su herencia es autosómica dominante. La frecuencia de la asociación es del 50-70% en las distintas series (AU)

Rhabdomyoma is a rare tumor usually detected by ultrasonography. This neoplasm represents approximately 1% of all intrauterine cardiac abnormalities. Development inside the uterus and the hemodynamic repercussions of this entity depend on tumoral growth. Large masses can cause obstructions and subsequently hydrops and tachyarrhythmias. In addition to these complications, rhabdomyoma is associated with tuberous sclerosis, which occupies an importance place in prenatal counseling since inheritance is autosomal dominant. The frequency of this association is between 50% and 70% in distinct series (AU)

Canine pyometra: a study of the urinary proteins by SDS-PAGE and Western blot.

Canine pyometra often causes glomerulonephritis by immune complex deposition in the glomeruli. Proteinuria, ranging from moderate to severe, may be present secondary to renal damage. To determine urinary protein excretion due to pyometra, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted on urine from 15 bitches with pyometra and 10 healthy bitches. To characterize urinary immunoglobin excretion, Western blot analysis of the urine samples using antibodies to canine IgG and IgA was also performed. Nine bands were detected by electrophoresis in bitches with pyometra, while only four were detected in the healthy animals. The urinary proteins from bitches with pyometra were primarily of glomerular origin; 58% were of medium-high molecular weight (MW), and the remainder were low MW. None of the healthy dogs had IgG or IgA in their urine, whereas three bitches with pyometra had IgG in their urine and another bitch with pyometra had both IgG and IgA. The low proportion of bitches with urinary immunoglobins was probably be due to early diagnosis of the disease. Although only a limited number of dogs was used, this study is apparently the first to characterize the electrophoretic pattern of urinary proteins and to quantify urinary excretion of IgG and IgA in bitches with pyometra.

Characterization of renal damage in canine leptospirosis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting of the urinary proteins.

Canine leptospirosis is a zoonotic disease that can cause interstitial nephritis. As a consequence of the renal damage, proteinuria may occur. To determine the urine protein pattern in the disease we performed sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on the urine from 10 dogs with leptospirosis and 20 healthy dogs. Western blotting analysis of the urine samples with antibodies against canine IgG and IgA was also performed to identify these immunoglobulins in the urine. Urine electrophoresis showed three new bands in the dogs suffering from leptospirosis. Only two of the dogs with leptospirosis showed detectable concentrations of IgG and IgA in urine, while a third animal showed IgG alone. The study showed a 36.7% increase in the excretion of low molecular weight proteins in dogs with leptospirosis but almost no change in the high molecular weight protein pattern. These results, together with the low number of animal with detectable concentrations of IgG and IgA in the urine, support the view that canine leptospirosis is characterized by interstitial nephritis.

Mechanisms of MPP(+) incorporation into cerebellar granule cells.

Exposure of cerebellar granule cells to 1-methyl-4-phenylpiridinium (MPP(+)) results in cell death. We have studied the implication of various membrane transporter systems on MPP(+) neurotoxicity, including the dopamine transporter system (DAT) and cationic amino acid transporters (CAT). We have showed a partial protection against MPP(+) toxicity when the dopamine transporter is inhibited by 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]4-(3-phenylpropyl)piperazinedihydrochloride (GBR-12909). However, almost full protection is only achieved by the simultaneous addition of GBR-12909 and cationic amino acids. These results suggest two ways system of MPP(+) entrance into cerebellar granule cells: the DAT with high activity and the CAT with low activity. We also demonstrated that 5,7-dichlorokynurenic acid (MK-801) failed to protect against MPP(+) exposure, evidencing that N-methyl-D-aspartate (NMDA) receptor is not involved in the MPP(+)-induced cell death.

Lithium inhibits caspase 3 activation and dephosphorylation of PKB and GSK3 induced by K+ deprivation in cerebellar granule cells.

Lithium protects cerebellar granule cells from apoptosis induced by low potassium, and also from other apoptotic stimuli. However, the precise mechanism by which this occurs is not understood. When cerebellar granule cells were switched to low potassium medium, the activation of caspase 3 was detected within 6 h, suggesting a role of caspase 3 in mediating apoptosis under conditions of low potassium. In the same conditions, lithium (5 mM) inhibited the activation of caspase 3 induced by low potassium. As lithium did not inhibit caspase 3 activity in vitro, these results suggest that this ion inhibits an upstream component that is required for caspase 3 activation. Lithium is known to inhibit a kinase termed glycogen sythase kinase 3 (GSK3), which is implicated in the survival pathway of phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB). Here we demonstrate that low potassium in the absence of lithium induces the dephosphorylation, and therefore the activation, of GSK3. However, when lithium was present, GSK3 remained phosphorylated at the same level as observed under conditions of high potassium. Low potassium induced the dephosphorylation and inactivation of PKB, whereas when lithium was present PKB was not dephosphorylated. Our results allow us to propose a new hypothesis about the action mechanism of lithium, this ion could inhibit a serine-threonine phosphatase induced by potassium deprivation.

Glu-256 is a main structural determinant for oligomerisation of human arginase I.

One determinant that could play a role in the quaternary structure of human arginase is the pair of salt links between the strictly conserved residues R255 from one monomer and E256 from every adjacent subunit. In this work, the ionic interaction between monomers was disrupted by expressing a human arginase where Glu-256 had been substituted by Gln. Biochemical analyses of the mutant protein showed that: (i) it shares the wild-type kinetic parameters of the arginine substrate; (ii) E256Q arginase behaves as a monomer by gel filtration; (iii) it is drastically inactivated by dialysis in the presence of EDTA, an inhibitory effect which is reversed by addition of Mn(2+); and (iv) the mutant enzyme loses thermal stability. The lack of oligomerisation for E256Q arginase and the conservation of E256 throughout evolution of the protein family suggest that this residue is involved in the quaternary structure of arginases.

Implications of the S-shaped domain in the quaternary structure of human arginase.

Arginase I is a homotrimeric protein with a binuclear manganese cluster. At the C-terminus of each monomer, the polypeptide chain forms an unusual S-shaped oligomerization motif where the majority of intermonomer contacts are located [Z.F. Kanyo, L.R. Scolnick, D.E. Ash, D.W. Christianson, Nature 383 (1996) 554-557]. In order to study the implication of this motif in the quaternary structure of human arginase I, we have constructed a truncated arginase lacking the 14 C-terminal amino acids, leaving Arg-308 as the last residue in the sequence. The resulting protein retains its trimeric structure, as determined by gel filtration (molecular mass 94 kDa). The same result was obtained in the presence of high ionic strength (KCl 0.5 M). Both data indicate that neither the S-shaped motif nor Arg-308 are fundamental in keeping the trimeric quaternary structure. Data obtained from intrinsic anisotropy and fluorescence intensity studies allow us to predict that the distance between the two unique tryptophans in the sequence is 2.9 nm in the native arginase and 4.1 nm for the truncated mutant. These distances allow us to assume a different conformational state in the truncated arginase without any change in its quaternary structure, suggesting that the carboxy-terminal motif is not the most prominent domain implicated in the quaternary structure of human arginase. Collisional quenching studies reinforce this possibility, since using I(-) as quenching molecule we were able to distinguish the two tryptophans in the truncated arginase. Moreover, kinetic studies show that the truncated mutant was fully active. In summary, the main conclusion about the structure of the human arginase I, derived from our study, is that the C-terminal S-shaped motif is not basic to the maintenance of the quaternary structure nor to the activity of the protein.

Different mechanisms of protection against apoptosis by valproate and Li+.

Acute treatment with valproate and Li+ was found to protect cultured cerebellar granule cells against apoptosis induced by low K+ (5 mM). Because the protection was unaffected by MK801 (N-methyl-D-aspartate receptor inhibitor), an increase in glutamate release cannot be responsible for the observed neuroprotection. Insulin also protects against low-K+-induced apoptosis of cerebellar granule cells. This protection is totally dependent on LY294002 (a phosphatidylinositol 3-kinase inhibitor). These results suggest a role for phosphatidylinositol 3-kinase in the neuroprotection induced by insulin. Likewise, and in contrast with the results observed with Li+, the protection induced by valproate is also dependent on insulin and LY294002. Moreover, valproate (a branched-chain fatty acid) does not change the plasma membrane microviscosity under physiological conditions. These results suggest that valproate protects against low-K+-induced apoptosis by acting in the phosphatidylinositol 3-kinase/protein kinase B pathway. The protection by Li+ is independent of this transduction pathway.

Th1/Th2-regulated expression of arginase isoforms in murine macrophages and dendritic cells.

Activated murine macrophages metabolize arginine by two alternative pathways involving the enzymes inducible NO synthase (iNOS) or arginase. The balance between the two enzymes is competitively regulated by Th1 and Th2 T helper cells via their secreted cytokines: Th1 cells induce iNOS, whereas Th2 cells induce arginase. Whereas the role of macrophages expressing iNOS as inflammatory cells is well established, the functional competence of macrophages expressing arginase remains a matter of speculation. Two isoforms of mammalian arginases exist, hepatic arginase I and extrahepatic arginase II. We investigated the regulation of arginase isoforms in murine bone marrow-derived macrophages (BMMPhi) in the context of Th1 and Th2 stimulation. Surprisingly, in the presence of either Th2 cytokines or Th2 cells, we observe a specific induction of the hepatic isoform arginase I in BMMPhi. Induction of arginase I was shown on the mRNA and protein levels and obeyed the recently demonstrated synergism among the Th2 cytokines IL-4 and IL-10. Arginase II was detectable in unstimulated BMMPhi and was not significantly modulated by Th1 or Th2 stimulation. Similar to murine BMMPhi, murine bone marrow-derived dendritic cells, as well as a dendritic cell line, up-regulated arginase I expression and arginase activity upon Th2 stimulation, whereas arginase II was never detected. In addition to revealing the unexpected expression of arginase I in the macrophage/monocyte lineage, these results uncover a further intriguing parallelism between iNOS and arginase: both have a constitutive and an inducible isoform, the latter regulated by the Th1/Th2 balance.

Partial lithium-associated protection against apoptosis induced by C2-ceramide in cerebellar granule neurons.

Primary cultures of cerebellar granule neurons, maintained in a serum-containing medium, underwent apoptosis when exposed to C2-ceramide, as assessed by mitochondrial reduction of MTT and intranucleosomal DNA fragmentation. After an 18 h exposure to 50 microM C2-ceramide, cell viability decreased by 25-40%. Addition of lithium together with C2-ceramide resulted in a partial protection of apoptosis, which was maximal at 5 mM lithium (37% protection). When lithium was added 5 h before the apoptotic stimulus the neuroprotective effect of the ion was clearly increased (66% protection). This effect was not due to intracellular inositol depletion or inhibition of NMDA receptors. Our data broaden the nature of apoptotic insults being reversed by lithium, stressing the neuroprotective effects of the ion.

The cytoplasmic loop between putative transmembrane segments 6 and 7 in sarcoplasmic reticulum Ca2+-ATPase binds Ca2+ and is functionally important.

Limited proteolysis by proteinase K of rabbit SERCA1 Ca2+-ATPase generates a number of fragments which have been identified recently. Here, we have focused on two proteolytic C-terminal fragments, p20C and p19C, starting at Gly-808 and Asp-818, respectively. The longer peptide p20C binds Ca2+, as deduced from changes in migration rate by SDS-polyacrylamide gel electrophoresis performed in the presence of Ca2+ as well as from labeling with 45Ca2+ in overlay experiments. In contrast, the shorter peptide p19C, a proteolysis fragment identical to p20C but for 10 amino acids missing at the N-terminal side, did not bind Ca2+ when submitted to the same experiments. Two cluster mutants of Ca2+-ATPase, D813A/D818A and D813A/D815A/D818A, expressed in the yeast Saccharomyces cerevisiae, were found to have a very low Ca2+-ATPase activity. Region 808-818 is thus essential for both Ca2+ binding and enzyme activity, in agreement with similar results recently reported for the homologous gastric H+, K+-ATPase (Swarts, H. G. P., Klaassen, C. H. W., de Boer, M., Fransen, J. A. M. , and De Pont, J. J. H. H. M. (1996) J. Biol. Chem. 271, 29764-29772). However, the accessibility of proteinase K to the peptidyl link between Leu-807 and Gly-808 clearly shows that the transmembrane segment M6 ends before region 808-818. It is remarkable that critical residues for enzyme activity are located in a cytoplasmic loop starting at Gly-808.

Expression of the sarcoplasmic reticulum Ca(2+)-ATPase in yeast.

We describe here an easy system for the production of mg amounts of the rabbit Ca(2+)-ATPase SERCA 1a in the yeast S. cerevisiae. The protein is present in several membranes, including the plasma membrane of the yeast, in a native conformation. It can be purified by immunoprecipitation and can be phosphorylated from ATP in a Ca(2+)-dependent manner. Using a temperature-sensitive secretion mutant strain, the fully active protein can also be obtained in secretory vesicles.

Sedimentation equilibrium of detergent-solubilized membrane proteins in the preparative ultracentrifuge.

Working with detergent-solubilized bacteriorhodopsin we have used a table top preparative centrifuge for determination of M(r) of membrane proteins by sedimentation equilibrium. We demonstrate the use of two new methods to measure protein concentration as a function of distance from rotor axis: (i) peak integration after HPLC on silica gel, and (ii) microdensitometry after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining. These methods, although somewhat lengthier than conventional spectrophotometric methods, are more reliable, especially in the presence of a large amount of detergent and small amount of protein. In addition they provide independent information on the status of the protein after sedimentation equilibrium, the association of the solubilized units being readily detected by gel chromatography and proteolytic cleavage by SDS-PAGE.

Differential scanning calorimetry study of glycogen phosphorylase b-detergent interactions.

The overall thermal denaturation of glycogen phosphorylase b is irreversible and our results conform to the theoretical prediction of a reversible process followed by a slower irreversible process. The basic thermodynamic parameters of glycogen phosphorylase b denaturation have been worked out and found to be: critical temperature 57.0 +/- 0.5 degrees C, transition half-width 8 +/- 1 degrees C, and calorimetric enthalpy change and Van't Hoff enthalpy change of the denaturation process 450 +/- 50 and 105 +/- 15 kcal/mol of enzyme monomer, respectively, at pH 7.4. These parameters have been found to be largely altered by the detergents octylglucoside, cholate, and deoxycholate at or below their critical micelle concentration, but not by Triton X-100 nor by lecithin liposomes. Organic solvents, such as dimethyl sulfoxide and methanol, and the presence of sarcoplasmic reticulum membranes produces an alteration of the denaturation thermogram of glycogen phosphorylase b similar to that produced by the above-mentioned detergents. These results allow us to hypothesize that hydrophobic domains of glycogen phosphorylase b are involved in its association to sarcoplasmic reticulum membranes in the sarcoplasmic reticulum/glycogenolytic complex of mammalian skeletal muscle.

Location of functional centers in the microsomal cytochrome P450 system.

Fluorescence quenching and energy-transfer studies have been carried out to determine the position of FAD and FMN groups of NADPH-cytochrome P450 reductase and of the heme and substrate groups of cytochrome P450 with respect to the lipid/water interphase. Quenching by iodine of the fluorescence of the flavins of the reductase shows a biphasic pattern, due to the different accessibility of FAD and FMN to the solvent with Stern-Volmer constants of 7.9 x 10(-4) and 2.7 x 10(-3) mM-1, respectively. Both prosthetic groups appear to be buried within the three-dimensional structure of the native reductase, FAD more deeply embedded than FMN and with a relative contribution to the total fluorescence of flavins of 84% (FAD) and 16% (FMN). The lack of significant energy transfer (less than 5%) from FAD+FMN to the rhodamine group of the N-labeled phosphatidylethanolamine incorporated in membranes reconstituted with NADPH-cytochrome P450 reductase and phosphatidylcholine points out that both groups are located at a distance greater than 5 nm from the lipid/water interphase. Steady-state fluorescence intensity and anisotropy data obtained with native and FMN-depleted NADPH-cytochrome P450 reductase show that energy transfer between both prosthetic groups occurs in the native reductase with an efficiency of ca. 31%, consistent with a separation between these groups of 2 nm as suggested earlier by Bastiaens, P. I. H., Bonants, P. J. M., Müller, F., & Visser, A. J. W. G. [(1989) Biochemistry 28, 8416-8425] from time-resolved fluorescence anisotropy measurements.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation by phosphorylation of glycogen phosphorylase-sarcoplasmic reticulum interaction.

Glycogen phosphorylase b at concentrations close to those found in skeletal muscle interacts with sarcoplasmic reticulum membranes, but not with liposomes made of lipids extracted from these membranes, and is inhibited upon binding to the membrane. The interaction of glycogen phosphorylase with the sarcoplasmic reticulum membrane is modulated by phosphorylation, for the a form of this enzyme shows a K0.5 of interaction about 10-fold lower than the b form. Upon association to the membrane the fluorescence properties of the coenzyme of glycogen phosphorylase, pyridoxal-5'-phosphate, are strongly altered, for the fluorescence at 535 nm is partially quenched and the fluorescence at 415-420 nm increases. Using fluorescein labeled sarcoplasmic reticulum membranes we have found that the average conformation of the Ca2+ + Mg(2+)-ATPase is also altered on binding of phosphorylase b. In conclusion, the results reported in this paper suggest that glycogen phosphorylase and Ca2+ + Mg(2+)-ATPase directly interact under experimental conditions similar to those found in the sarcoplasm, and that this interaction is modulated by phosphorylation of the phosphorylase.

[The recurrence of duodenal ulcer in patients undergoing treatment with ranitidine and placebo. A multicenter study]. / Recurrencia de la úlcera duodenal en pacientes sometidos a tratamiento con ranitidina y placebo. Estudio multicéntrico.

This randomised, double-blind multicenter study was conducted in order to evaluate the long-term effect (one year) of 150 mg ranitidine vs placebo in 51 patients with healed duodenal ulcer. Seventeen patients had ulcer recurrence at the end of follow-up, one among the 24 patients that received ranitidine and 16 among the 27 patients that received placebo (p = 0.00009). No side effects was detected. Our results support the usefulness of a one-year maintenance therapy with 150 mg ranitidine.