Development and validation of a clinical score for early diagnosis of constipation in critically ill children.

Constipation affects almost 50% of critically ill pediatric patients and is related to their morbidity and mortality. However, little attention is paid to it and it is diagnosed late and when there are already complications. The objective of this study is to develop and validate a score to identify critically ill children with high risk of constipation 48 h after admission. A single center two phase-study was carried out; the first one (retrospective observational study) to develop the score and the second one to validate it in another prospective observational study. Children between 15 days of life and 18 years old admitted to the PICU for more than 3 days were included. Demographic and clinical data during the first 48 h after PICU admission were collected. Univariate and multivariate analysis and ROC curves were used to develop and validate the score. Data from 145 patients (62.8% boys) with a mean age of 34.9 ± 7.3 months were used to develop the score. Independent factors identified to develop the score were: weight > 7 kg, admission to PICU after surgery, need of vasoconstrictors, doses of fentanyl ≥ 2 mcg/kg/h, and initiation of enteral nutrition later than 48 h after admission. Two cut-off values were identified to set low constipation risk (< 5.7 points) and high constipation risk (> 6.2 points). This score was validated in 124 patients showing a sensibility of 63.2%, specificity of 95.5% and a positive/negative predictive values (P/NPV) of 100% and 82.1% respectively to identify constipated patients. This is the first score to identify high constipation risk in critically ill children. This score is easy to apply, and internal validation has shown a PPV of 100%.

Changes in hemodynamics, renal blood flow and urine output during continuous renal replacement therapies.

Continuous renal replacement therapies (CRRT) affect hemodynamics and urine output. Some theories suggest a reduced renal blood flow as the cause of the decreased urine output, but the exact mechanisms remain unclear. A prospective experimental study was carried out in 32 piglets (2-3 months old) in order to compare the impact of CRRT on hemodynamics, renal perfusion, urine output and renal function in healthy animals and in those with non-oliguric acute kidney injury (AKI). CRRT was started according to our clinical protocol, with an initial blood flow of 20 ml/min, with 10 ml/min increases every minute until a goal flow of 5 ml/kg/min. Heart rate, blood pressure, central venous pressure, cardiac output, renal blood flow and urine output were registered at baseline and during the first 6 h of CRRT. Blood and urine samples were drawn at baseline and after 2 and 6 h of therapy. Blood pressure, cardiac index and urine output significantly decreased after starting CRRT in all piglets. Renal blood flow, however, steadily increased throughout the study. Cisplatin piglets had lower cardiac index, higher vascular resistance, lower renal blood flow and lower urine output than control piglets. Plasma levels of ADH and urine levels of aquaporin-2 were lower, whereas kidney injury biomarkers were higher in the cisplatin group of piglets. According to our findings, a reduced renal blood flow doesn't seem to be the cause of the decrease in urine output after starting CRRT.

Is Hydrogen Breath Test with Lactulose Feasible for Measuring Gastrocecal Transit in Critically Ill Children? Pilot Study about Modification of the Technique.

Introduction. Gastrocecal transit time (GCTT) can be measured by exhaled hydrogen after lactulose intake (lactulose-eH2 test). The objectives were to assess whether it is possible to carry out this test in critically ill children with and without mechanical ventilation (MV) and to analyze whether the results are consistent with clinical findings. Methods. Patients admitted to the Pediatric Intensive Care Unit (PICU) for more than 3 days were included. Those with gastrointestinal disease prior to admission were excluded. A modified technique to obtain eH2 from the ventilator tubes was performed. Results. Sixteen patients (37.5% boys) with a median age of 19 (5-86.5) months were included. Five patients (31.2%) were breathing spontaneously but lactulose-eH2 test could not be performed while it could be performed successfully in the 11 patients with MV. Seven patients (63.3%) did not show an eH2 peak. The other 4 showed a median time of 130 min (78.7-278.7 min) from lactulose intake to a 10 ppm eH2 peak. Children with an eH2 peak had intestinal movements earlier [6.5 (1.5-38.5) versus 44 (24-72) hours p = 0.545]. Conclusion. Although the designed adaption is useful for collecting breath samples, lactulose-eH2 test may not be useful for measuring GCTT in critically ill children.

Pallial oviduct of Pomacea canaliculata (Gastropoda): ultrastructural studies of the parenchymal cellular types involved in the metabolism of perivitellins.

Seasonal variations in the morphology of the parenchymal mass and function of the albumen gland/capsule gland complex have been studied in Pomacea canaliculata, together with the cellular types involved in the synthesis and secretion of perivitellin fluid components. The two major parenchymal cell types, albumen secretory cells (AS) and labyrinthic cells (LC), undergo seasonal variations throughout the annual reproductive cycle, which is divided into three periods. Both cellular types show maximal development and structural complexity during the reproductive period (spring and summer). AS cells have a well-developed Golgi complex and rough endoplasmic reticulum and their secretory granules show electron-dense particles of about 20 nm (probably galactogen). These cells are uniquely involved in ovorubin and PV2 perivitellin synthesis and their secretory granules are the single storage site for these two major perivitellins, as revealed by immunoelectron microscopy. AS also possess calcium deposits that infiltrate the cytoplasmic matrix. The luminal surfaces of LC exhibit long cilia intermingled with sparce short microvilli. Basally, the plasma membrane shows deep irregular folds that extend through the cytoplasm up to the subapical region. Calcium deposits infiltrate the cytoplasm and accumulate in the extracellular space of the basal labyrinth. Nerve terminals seem to be involved in the regulation of parenchymal cell secretion. At the post-reproductive period, AS markedly change their aspect following the release of most of the secretory granules into the acinar lumen. LC decrease in volume, the number of their cilia decreases, their cytoplasmic folds are much thinner and their extracellular spaces lack calcium particles. At the pre-reproductive period (winter), AS and LC recover and prepare for the subsequent period.

Involvement of blood serum in amphibian fertilization

A proteolytic enzyme secreted by the first portion of amphibian oviduct, pars recta, called oviductin in Xenopus laevis, causes ultrastructural alterations on the extracellular matrix of coelomic eggs, turning them susceptible to fertilization. Although great advances have been made in the field of reproduction, the molecular mechanisms responsible for the fusion between the egg and the sperm are yet to be understood. We have recently demonstrated the presence of proteins from pars recta fluid in blood serum and extracellular matrix of coelomic eggs in Bufo arenarum. Here we show, using immunofluorescence procedures, that blood serum components are present in the extracellular matrix of coelomic and pars recta fluid-conditioned eggs in Bufo arenarum. Furthermore, by assessing the neutralizing effect on the conditioning activity of pars recta fluid on coelomic eggs we found that antibodies against pars recta secretions and blood serum inhibited the effect of sperm-lysin on the vitelline envelope of conditioned oocytes and impaired fertilization by sperm. Thus, serum proteins appear to be implicated in the molecular events that lead to amphibian fertilization

Involvement of blood serum in amphibian fertilization

A proteolytic enzyme secreted by the first portion of amphibian oviduct, pars recta, called oviductin in Xenopus laevis, causes ultrastructural alterations on the extracellular matrix of coelomic eggs, turning them susceptible to fertilization. Although great advances have been made in the field of reproduction, the molecular mechanisms responsible for the fusion between the egg and the sperm are yet to be understood. We have recently demonstrated the presence of proteins from pars recta fluid in blood serum and extracellular matrix of coelomic eggs in Bufo arenarum. Here we show, using immunofluorescence procedures, that blood serum components are present in the extracellular matrix of coelomic and pars recta fluid-conditioned eggs in Bufo arenarum. Furthermore, by assessing the neutralizing effect on the conditioning activity of pars recta fluid on coelomic eggs we found that antibodies against pars recta secretions and blood serum inhibited the effect of sperm-lysin on the vitelline envelope of conditioned oocytes and impaired fertilization by sperm. Thus, serum proteins appear to be implicated in the molecular events that lead to amphibian fertilization

Involvement of blood serum in amphibian fertilization.

A proteolytic enzyme secreted by the first portion of amphibian oviduct, pars recta, called oviductin in Xenopus laevis, causes ultrastructural alterations on the extracellular matrix of coelomic eggs, turning them susceptible to fertilization. Although great advances have been made in the field of reproduction, the molecular mechanisms responsible for the fusion between the egg and the sperm are yet to be understood. We have recently demonstrated the presence of proteins from pars recta fluid in blood serum and extracellular matrix of coelomic eggs in Bufo arenarum. Here we show, using immunofluorescence procedures, that blood serum components are present in the extracellular matrix of coelomic and pars recta fluid-conditioned eggs in Bufo arenarum. Furthermore, by assessing the neutralizing effect on the conditioning activity of pars recta fluid on coelomic eggs we found that antibodies against pars recta secretions and blood serum inhibited the effect of sperm-lysin on the vitelline envelope of conditioned oocytes and impaired fertilization by sperm. Thus, serum proteins appear to be implicated in the molecular events that lead to amphibian fertilization.

Characterization and properties of steroid binding protein in Bufo arenarum serum.

Serum steroid binding properties of mature Bufo arenarum females were studied. Binding data obtained using charcoal adsorption assay and equilibrium dialysis methods indicates a single protein, named Bufo arenarum sex binding protein (Ba SBP), which binds 5 alpha-dihydrotestosterone (DHT), testosterone (T), and estradiol-17 beta (E2) with high affinity (10(-7) M-1 - 10(8) M-1) and fair capacity (10(-6) M). Scatchard plot analysis demonstrated the coexistence of two binding sites. Ba SBP has a sedimentation coefficient of 5.2 S in sucrose gradient centrifugation in low salt and under steady-state conditions. The specificity of this protein, determined by competitive binding experiments, is comparable to human SBP. DHT and T bind with higher affinity than E2. Estriol and estrone competed poorly, while diethylstilbestrol and C21 steroids did not compete. The binding capacity of this protein is under estrogenic control.

In vivo and in vitro protein synthesis of oviducal pars recta by estradiol effect.

1. Proteins secreted by toad oviductal pars recta are involved in fertilization and their biological activity is regulated by estrogens. 2. Effect of 17 beta-estradiol on protein synthesis was examined on castrated animals by different in vivo and in vitro experimental approaches. 3. Different routes of L-[3H]leucine administration were assessed. An intralymphatic route was the most efficient for incorporating radioactivity per mg of trichloroacetic acid insoluble proteins. 4. Ligature of pars recta induces protein synthesis at a similar level to exogenous estradiol. 5. Electrophoretic pattern of radioactive proteins did not show synthesis of a specific protein related to the zone with biological activity. 6. Pars recta releases newly synthesized proteins in vivo into its fluid secretion as much as in vitro into culture medium.

Properties of an oviducal protein involved in amphibian oocyte fertilization.

The secretion produced at the most cephalic portion of the oviduct of the toad (pars recta in Bufo arenarum) is involved in fertilization. Although the present study indicates that a prerequisite for the fertilization of coelomic eggs is either their passage through the pars recta (PR) or their treatment with PR secretion fluid prior to insemination, the exact role of this secretion in the fertilization process is still not clearly understood. A protein acting upon the vitelline envelope (VE) of Bufo arenarum coelomic eggs has been purified from secretion fluid (pars recta protein, PRP). Some properties of both this protein and the secretion fluid are examined in an effort to understand their mechanism of action. It was shown that PRP partially dissolves the VE of coelomic oocytes in a way that resembles the action of proteolytic enzymes. PRP has also proved to be active on synthetic substrates of proteolytic enzymes such as p-Tosyl-L-arginine methyl ester-HCl (TAME), alpha-N-benzoyl-L-arginine ethyl ester-HCl (BAEE), and alpha-N-benzoyl-DL-arginine-p-nitroanilide HCl (BAPNA). PR enzyme is activated by calcium ions, shows a broad peak of maximum activity at pH 7.8, is stable at alkaline pH, and is inhibited by soybean trypsin inhibitor (SBTI) and N-alpha-p-tosyl-L-lysine chloromethyl ketone HCl (TLCK) but not by lima bean trypsin inhibitor (LBTI) or L-1-tosyl-amide-2-phenylethylchloro-methyl-ketone (TPCK).

A trypsin-like oviducal proteinase involved in Bufo arenarum fertilization.

When the jelly-less eggs removed from the most cephalic region of the oviduct (pars recta) of the toad Bufo arenarum were inseminated at a high sperm concentration, high frequencies of fertilization were obtained. On the other hand, control eggs removed from the pleuroperitoneal cavity (coelomic eggs)) were neither fertilized upon insemination under identical conditions, nor with the water extract of the jelly. Under these inseminating conditions, however, a high frequency of fertilization was obtained when coelomic eggs were preincubated in the presence of the fluid secreted by the epithelium of the pars recta or of an extract prepared from pars recta homogenate. Experimental evidence is presented showing that the component responsible for this effect acts on the vitelline envelope of the egg, increasing its susceptibility to sperm lysin. It is probable, therefore, that it induces successful fertilization of coelomic eggs by making the vitelline envelope more easily penetrable by sperm. The active factor was partially purified by Sephadex chromatography. The product obtained was of high activity and, as judged by its inhibition with soybean trypsin inhibitor and lima-bean trypsin inhibitor, it is likely to be a trypsin-like enzyme. The molecular weight of the factor was estimated to be 47000 by Sephadex chromatography. Secretion of the pars recta factor is hypophysis-dependent and its activity is not influenced by pH within the range testes (6.0--8.4).

An oviducal enzyme isolated by affinity chromatography which acts upon the vitelline envelope of Bufo arenarum coelomic oocytes.

A trypsin-like oviducal proteinase acting upon the vitelline envelope of Bufo arenarum coelomic oocytes has been purified to apparent homogeneity by gel filtration on Sephadex G-200 and by affinity chromatography on a column of Sepharose 4-B containing covalently bound concanavlin A (Con A). The biologically active molecule migrated as a single band of protein upon SDS polyacrylamide gel electrophoresis.