[Rotavirus laboratory network: results after one year of observation]. / Red de laboratorios de Rotavirus: resultados del primer año de vigilancia.

Rotavirus is the most common cause of severe diarrhea in children and it has been estimated that in Argentina Rotavirus is responsible for 21,000 hospitalizations, 85,000 medical attentions and an annual medical cost of US$ 27 millions. Given that a Rotavirus vaccine is about to be approved, a laboratory network based surveillance system was organized. Herein, we present the results after one year of study. Severe diarrhea was responsible for 9% of pediatric hospitalizations and rotavirus was detected in 42.1% of the diarrhea cases. We estimated that Rotavirus causes 3.8% of pediatric hospitalizations. The number of diarrhea and Rotavirus diarrhea hospitalizations was greater during the first year of life (62% and 71.3%, respectively). The number of diarrhea hospitalizations during the December-May semester was significantly higher than the rest of the year. A Rotavirus diarrhea peak was detected between April and June. These results indicate that Rotavirus is the most important etiological agent of severe diarrhea in Argentine children and show the importance of performing Rotavirus diagnosis in every pediatric hospital. The additional costs will be compensated by many benefits such as better use of antibiotics, improved nosocomial spread control, better handling of hospital beds and of laboratory resources and of the hospitalized patient.

ATP dependence of Na+/H+ exchange. Nucleotide specificity and assessment of the role of phospholipids.

We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na+/H+ antiporter, using the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pHi) by microfluorimetry. Na+/H+ exchange activity was measured as the Na(+)-driven pHi recovery from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably transfected with NHE-1, omission of ATP from the pipette solution inhibited Na+/H+ exchange. Conversely, ATP perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-D-glucose and oligomycin. In cells dialyzed in the presence of ATP, no "run-down" was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of the antiporter was obtained at approximately 5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na+/H+ exchange even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca2+ exchanger, this requirement has been attributed to an aminophospholipid translocase, or "flippase.". The involvement of this enzyme in Na+/H+ exchange was examined using fluorescent phosphatidylserine, which is actively translocated by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na+/H+ exchange, implying that the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATP gamma S were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted for ATP. In contrast, GTP gamma S was ineffective. We conclude that ATP is the only soluble factor necessary for optimal activity of the NHE-1 isoform of the antiporter. Mg2+ does not appear to be essential for the stimulatory effect of ATP. We propose that two mechanisms mediate the activation of the antiporter by ATP: one requires hydrolysis and is likely an energy-dependent event. The second process does not involve hydrolysis of the gamma-phosphate, excluding mediation by protein or lipid kinases. We suggest that this effect is due to binding of ATP to an as yet unidentified, nondiffusible effector that activates the antiporter.

Assessment of the contribution of the cytochrome b moiety of the NADPH oxidase to the transmembrane H+ conductance of leukocytes.

Phagocytic cells can kill microorganisms by synthesizing superoxide. Activation of the NADPH oxidase that generates superoxide is accompanied by a large intracellular burst of metabolic acid production. Despite the excess acid generation, cytosolic pH (pHi) remains near neutrality due to the concomitant stimulation of several homeostatic H+ extrusion mechanisms including a recently described H(+)-conductive pathway. Activation of the conductance by phorbol esters is defective in neutrophils of chronic granulomatous disease (CGD) patients lacking the transmembrane cytochrome b subunits of the NADPH oxidase. This finding suggests that the oxidase itself undertakes H+ translocation or that, alternatively, assembly of the oxidase is required to activate a separate H+ conducting entity. To distinguish between these possibilities, the presence of the conductive pathway was assessed in unstimulated normal and CGD cells by manipulating pHi and the transmembrane potential. Using fluorimetric determinations of pHi, a conductive, Zn(2+)-sensitive alkalinization was observed in neutrophils from both normal and cytochrome b-deficient CGD donors. The electrophysiological properties of the conductance were defined in purified blood monocytes using the whole cell configuration of the patch clamp. Depolarizing pulses induced slowly activating outward currents in cells from both normal and cytochrome b-deficient individuals. The elicited currents were potentiated by cytosolic acidification and did not inactivate within the times tested. As in control leukocytes, the reversal potential of tail currents in the CGD cells closely approximated the H+ equilibrium potential and was unaffected by substitution of the major ionic components of the external bathing medium. At all voltages tested, the magnitude of the evoked currents was comparable in normal and CGD cells. The results indicate that, like macrophages and granulocytes, human monocytes display a voltage-gated highly H(+)-selective conductance. More importantly, our findings imply that the conductive pathway is present in cells devoid of cytochrome b. Therefore, the defective activation of the conductive pathway by protein kinase C agonists in CGD cells is not due to the physical absence of the transporter. Instead we propose that the oxidase functions in a regulatory capacity, facilitating the opening of a distinct H+ conductance during cellular stimulation.

Focal localization of the NHE-1 isoform of the Na+/H+ antiport: assessment of effects on intracellular pH.

Na+/H+ exchange (antiport) is a major pathway for the regulation of intracellular pH. Antiport activity is stimulated when suspended cells adhere to the substratum. In this report, immunofluorescence was used to study the subcellular localization of the ubiquitous NHE-1 isoform of the antiport. NHE-1 was not distributed homogeneously on the surface of the cells. Instead, antiports were found to accumulate along the border of lamellipodia and near the edge of finer processes. Dual immunofluorescence experiments demonstrated that vinculin, talin and F-actin are concentrated at sites of NHE-1 accumulation. A mutated construct of NHE-1 lacking residues 566-635 of the cytosolic domain also accumulated near marginal lamellae. In contrast, the focal distribution observed in adherent cells was not detectable in cells grown in suspension. Fluorescence ratio imaging was used to define the functional consequences of focal accumulation of NHE-1. In the steady state, the pH was virtually identical throughout the cytosol. Moreover, no pH gradients were found to develop when cells recovered from an acid load by activation of Na+/H+ exchange. This is probably because of the presence of high concentrations of mobile buffers in the cytosol. The focal accumulation of antiporters near the cell margins may be involved in stimulation by adherence and/or generation of local osmotic gradients.

Determinants of the phagosomal pH in macrophages. In situ assessment of vacuolar H(+)-ATPase activity, counterion conductance, and H+ "leak".

We studied the factors that determine the intraphagosomal pH (pHp) in elicited murine peritoneal macrophages. pHp was measured in situ by recording the fluorescence of covalently fluoresceinated Staphylococcus aureus ingested by the macrophages. Following spontaneous acidification of the phagosomes, passive (leak) H+ permeability was determined measuring the rate of change of pHp upon complete inhibition of the H+ pump with bafilomycin A1. A significant, but comparatively low passive H+ permeability was detected. The existence of a passive H+ leak implies that continuous energy expenditure is required for the maintenance of an acidic pHp. In combination with ionophores, bafilomycin was also used to estimate the counterion permeability. The counterion conductance was found to be severalfold higher than the H+ leak. Ion substitution experiments in electropermeabilized cells and the inhibitory effects of quinine and 5-nitro-2-(3-phenylpropylamino)benzoic acid suggest that both monovalent anions and cations permeate the phagosomal membrane. The activity of the H+ pump was measured at various pHp levels. In the steady state, the rate of H+ pumping was considerably lower than counterion permeation. These findings suggest that the phagosomal membrane potential is insignificant. Consistent with this notion, increasing phagosomal conductance with ionophores failed to accelerate the rate of H+ pumping. Thus, the transmembrane delta pH is the predominant component of the proton-motive force across the phagosomal membrane in the steady state. The rate of H+ pumping was found to decrease steeply as the phagosomal lumen became acidified. Therefore, the pH sensitivity of the H+ pump, which possibly reflects a kinetic or allosteric effect, is the primary determinant of pHp.

Assessment of Na+-H+ exchange activity in phagosomal membranes of human neutrophils.

To assess the presence of Na+-H+ exchange in internalized membranes, the phagosomal pH was monitored in suspensions of intact human neutrophils by measuring the fluorescence of ingested bacteria (Micrococcus lysodeikticus) prelabeled with a pH-sensitive dye. Uptake of fluoresceinated bacteria was confirmed by flow cytometry and by phase-contrast and electron microscopy. Manipulation of the cytoplasmic ion content was accomplished by permeabilization of the plasma membrane with nystatin, which did not alter phagosomal permeability. At 37 degrees C, the phagosomal interior acidified at a maximal rate of 0.135 +/- 0.003 pH units/min (n = 10). The endogenous Na+-H+ exchanger does not affect phagosomal acidification, since the rate and extent of the pH change were not altered by 1) omission of intraphagosomal Na+ and 2) addition of the permeant inhibitor methylisobutylamiloride or by trapping amiloride in the phagosome during bacterial ingestion. Moreover, amiloride-sensitive Na+-H+ exchange was not detectable when Na+ or H+ gradients were imposed across the phagosomal membrane. Under comparable conditions, Na+-H+ exchange could be readily detected across the surface membrane. These data imply that the Na+-H+ antiporters are either inactivated in the phagosome or are segregated and not internalized into the phagosomal membrane.

[Counterimmunoelectrophoresis in the diagnosis of Bordetella pertussis]. / Contrainmunoelectroforesis para el diagnóstico de Bordetella pertussis.

The serum of 50 patients between 25 days to 15 years old who were admitted with the diagnosis of pertussis syndrome were investigated for precipitating antibodies by means of discontinuous counterimmunolectrophoresis (CIED). The presence of antibodies for Bordetella pertussis antigen was shown in 28 cases. All samples were taken within the first 24/48 hours of admission and at convalescence. The technique is easy to carry out, quick and of low cost. This test offers an adequate and fast means to differentiate a Bordetella pertussis syndrome form others produced by different viral etiologies. Besides, this serologic technique shows earlier results and of lower cost than classic techniques such as agglutination and complement fixation which are more difficult to apply.