Evaluation of Subclinical Fluid Overload Using Lung Ultrasound and Estimated Plasma Volume in the Postoperative Period Following Kidney Transplantation.

AIM: B-lines count measured with lung ultrasound (LUS) quantifies extravascular lung water and is validated in the setting of acute cardiac failure or chronic dialysis. Patients are often kept in moderately overhydrated states during the early postoperative period following kidney transplantation (KT). We described congestion changes during the early postoperative period following KT and the feasibility of LUS in this setting. METHODS: LUS (28 scanning-points method) and inferior vena cava (IVC) measurements were routinely performed in 36 patients after KT. Estimated plasma volume (ePV) was calculated from hemoglobin and hematocrit levels. RESULTS: No patient had >15 B-lines during the hospital stay. B-lines slightly increased until Day 4 after KT (Day 1, 1.7 ± 1.7; Day 4, 2.5 ± 2.5) and decreased up to Day 10 (1.4 ± 2.2; P vs Day 4 <.05). More B-lines were observed in patients aged older than 60 (P = .01 at Day 4) whereas IVC diameter and ePV were similar. In patients older than 60, B-lines had weak correlation with body weight variation (r = 0.64; P < .05), IVC diameters (r = 0.59 at Day 4 and r = 0.58 at Day 10; P < .05) but a strong correlation with ePV (r = 0.93 at Day 14; P < .05). B-line changes from Day 1 to Day 10 correlated with IVC diameter changes (r = 0.62; P < .05). CONCLUSION: LUS identifies subtle congestion changes during the early postoperative period following KT. The hyperhydration strategy usually followed during this period does not result in overt pulmonary congestion as assessed by LUS, even in older recipients.

Antidote Dilemma--an activity to promote critical thinking.

BACKGROUND: In a health care environment of increased patient acuity and greater demands on nursing practitioners, the ability to think critically is requisite to functioning safely and proficiently. METHOD: Definitions and tenets of critical thinking promulgated by Brookfield (1987) and Paul (1992) provided the theoretical foundation for Antidote Dilemma, an activity designed to promote critical thinking. RESULTS: Nurses who participated in the activity identified plausible solutions to a nursing dilemma, considered context in identifying alternatives, and thought about their thinking. CONCLUSION: Critical thinking is most likely to occur and continue when it is supported by others and repeatedly practiced. Continuing/staff-development educators play a vital role in promoting critical thinking among nursing practitioners.

Research utilization and the continuing/staff development educator.

Current health care imperatives and the continuous explosion of information and technology present new challenges for continuing/staff development educators as the nursing profession reinvents itself during the current crisis in health care. This turbulent health care environment necessitates utilization of current research findings to maintain the continued efficient and effective functioning of nursing staff. Familiarity with recent research utilization projects and models and barriers perceived to impede research utilization can assist the continuing/staff development educator to bridge the gap between knowledge generation and knowledge utilization.

Measurement strategies: the visual analogue scale.

Measuring psychosocial responses to health problems poses a unique challenge for the clinician searching for empirical indicators of these abstract constructs. Subjective phenomena such as pain, craving, or well-being vary in levels of intensity and are often difficult for the individual to describe in concrete terms. Visual analogue scales provide a valid and reliable solution to this challenging measurement problem.

Exploring the validity of data-gathering instruments.

The validity of an instrument is another important issue for the clinician to consider when selecting a tool for use in data collection. Broadly defined, validity refers to the extent to which an instrument measures what it is supposed to be measuring. Although validity is a unitary concept, this article explores three common categories of validity: content, criterion-related, and construct validity.

Assessing and enhancing reliability.

When choosing a tool or instrument to gather data, the clinician must consider many factors including the validity, reliability, sensitivity, and specificity of the measuring device. While all of these factors are important measurement issues, this article focuses on assessment and enhancement of reliability.

Synthesis of research findings through meta-analysis.

Meta-analysis is an alternative, quantitative approach to the analysis and synthesis of multiple investigations of the same clinical question. The statistical approaches that have been developed since Glass coined the term meta-analysis in 1976 are discussed. Clinicians will find meta-analysis helpful when there are conflicting research findings about perplexing clinical problems.

Tissue interface pressure and estimated subcutaneous pressures of 11 different pressure-reducing support surfaces.

This pilot study examined the pressure-reducing properties of 11 different pressure-reducing devices as compared to a standard hospital mattress. Mean trochanteric and heel pressure readings were obtained on each surface from 13 healthy adult volunteers by using an electropneumatic pressure transducer (Gaymar, catalog # PSM1). Mean trochanteric pressures ranged from 37.2 mm Hg to 55.1 mm Hg on the pressure-reducing support surfaces as compared to 83.6 mm Hg on a standard hospital mattress. Mean heel pressure readings ranged from 28.1 mm Hg to 62.1 mm Hg on the pressure-reducing support surfaces as compared to 93.9 mm Hg on the standard hospital mattress. While pressure-reducing support surfaces were found to yield significantly lower mean pressure readings than the standard hospital mattress, none of them is capable of preventing tissue ischemia if the subcutaneous pressure is three to five times higher than the interface pressure.

Purification and amino-terminal sequence of an insulin-like growth factor-binding protein secreted by rat liver BRL-3A cells.

A protein preparation that specifically binds insulin-like growth factors (IGFs) I and II was purified from medium conditioned by rat liver BRL-3A cells using molecular sieve chromatography in 1 M acetic acid followed by affinity chromatography on IGF-II-agarose. The affinity-purified IGF-binding protein exhibits a single major band with apparent Mr = 36,300 under reducing conditions on sodium dodecyl sulfate-polyacrylamide gels. The IGF-binding protein is efficiently and specifically cross-linked to either 125I-IGF-I (human) or 125I-IGF-II (rat) using disuccinimidyl suberate. An IGF-binding protein of similar apparent molecular weight was also affinity purified from rat hepatoma H-35 cell conditioned medium and found to differ from the BRL-3A protein such that potent polyclonal antisera prepared in rabbits against the purified BRL-3A IGF-binding protein exhibited a much lower titer for the H-35 protein in an enzyme-linked immunosorbent assay and upon immunoblotting. In order to determine whether a single BRL-3A IGF-binding protein is present in the affinity-purified preparation, the protein was prepared for sequencing on a Sephacryl S-300 column in 6 M guanidine HCl after reduction and alkylation. The amino acid composition (expressed in percentages) of this IGF-binding protein was determined to be: Cys = 5.5, Lys = 4.8, His = 2.8, Arg = 7.8, Asx = 10.2, Thr = 5.1, Ser = 3.9, Glx = 15.7, Gly = 17.4, Ala = 7.3, Val = 4.6, Met = 1.4, Ile = 2.4, Leu = 8.3, Tyr = 1.0, Phe = 1.9. Sequencing of the NH2-terminal portion of this protein led to the identification of 31 amino acids in the following order: Phe-Arg-Cys-Pro-Pro-Cys-Thr-Pro-Glu-Arg-Leu-Ala-Ala-Cys-Gly-Pro-Pro-Pro- Asp-Ala-Pro-Cys-Ala-Glu-Leu-Val-Arg-Glu-Pro-Gly-Cys. We conclude that rat liver BRL-3A cells secrete a single major IGF-binding protein capable of binding both IGF-I and IGF-II.

The insulin-like growth factor II receptor is phosphorylated by a tyrosine kinase in adipocyte plasma membranes.

Incorporation of 32P from [gamma-32P]ATP into tyrosine residues of the insulin-like growth factor (IGF)-II receptor was observed in a Triton X-100-insoluble fraction of rat adipocyte plasma membranes. IGF-II receptor phosphorylation proceeded to a stoichiometry of approximately 0.5 mol of phosphate/IGF-II binding site after 10 min of incubation at 4 degrees C. A Km for ATP of 6 microM was calculated for this phosphorylation reaction. Addition of IGF-II caused an approximately 2-fold increase in tyrosine phosphorylation of the IGF-II receptor in this preparation. In contrast, phosphorylation of angiotensin II by the Triton X-100 washed membranes was not stimulated by IGF-II. Incubation of purified receptor immobilized on IGF-II agarose or of receptor-enriched low density microsomal membranes with [gamma-32P]ATP did not result in appreciable incorporation of [32P]phosphate into the IGF-II receptor nor into exogenous substrates. These data suggest that the IGF-II receptor is not a tyrosine protein kinase capable of autophosphorylation but that it is a substrate for a tyrosine protein kinase endogenous to the adipocyte plasma membrane. The stimulatory effect of IGF-II on the tyrosine phosphorylation of its receptor may be due to a conformational change which converts the receptor to a better substrate for this tyrosine kinase.

C5a fragment of bovine complement. Purification, bioassays, amino-acid sequence and other structural studies.

C5a and des-Arg-C5a have been purified from bovine serum in milligram amounts. The progress of the purification was followed by measuring the chemotactic activity of the complement fragments. The two polypeptides induce activation of neutrophil-oriented locomotion and secretion with very similar dose/response effects. After preparing a rabbit antiserum to bovine C5a/des-Arg-C5a, a competitive enzyme-linked immunosorbent assay (ELISA) was set up for the detection of C5a from 5 ng/mol to 1 microgram/ml. The complete primary structure of bovine C5a, which consists of 74 amino acids, has been determined by sequence analysis of the tryptic peptides, aligned by peptides derived from a chymotryptic digest, and by partially sequencing the intact molecule. Bovine C5a has a sequence homology of 78% and 70% with porcine and human C5a, respectively, reacts with an antiserum to porcine C5a and is recognized by cell surface receptors on human neutrophils. Finally, the secondary structure of bovine C5a was investigated by circular dicroic spectroscopy and predicted from the amino acid sequence. A comparison of the content and distribution of alpha-helical and/or hydropathic regions, suggests that the three-dimensional structure of C5a might be modeled from the known crystal structure of the homologous C3a molecule.

Stimulation by insulin-like growth factors is required for cellular transformation by type beta transforming growth factor.

Medium conditioned by BRL-3A cells, a known source of insulin-like growth factor II (IGF-II), induced phenotypic transformation (anchorage-independent proliferation) of mouse BALB/c 3T3 fibroblasts but not rat NRK-49F fibroblasts, in the presence of 10% calf serum. A specific radioreceptor assay and a bioassay indicated that BRL-3A conditioned medium contained 0.5-1 ng/ml of type beta transforming growth factor (beta TGF). Purified IGF-II and beta TGF acting together reconstituted the transforming activity of BRL-3A conditioned medium on BALB/c 3T3 cells. Insulin was 5-10% as potent as IGF-II in supporting the transforming action of beta TGF on BALB/c 3T3 cells. NRK-49F cells were phenotypically transformed by beta TGF in the presence of EGF and 10% calf serum as the sole source of IGFs. However, transformation of NRK-49F cells under these conditions was inhibited by addition of purified IGF-binding protein. Addition of an excess of IGF-II prevented the inhibitory action of IGF-binding protein. The different sensitivity of the two cell lines to IGFs was correlated with lower levels of type I IGF receptor and higher levels of type II IGF receptor in NRK-49F cells as compared with BALB/c 3T3 cells. The results suggest that cellular stimulation by IGFs is a prerequisite for transformation of rodent fibroblasts by beta TGF. We propose that transformation of fibroblasts by beta TGF requires concomitant stimulation by the set of growth factors that support normal cell proliferation.

The type II insulin-like growth factor receptor does not mediate increased DNA synthesis in H-35 hepatoma cells.

The immunoglobulin fraction prepared from the serum of a rabbit immunized with purified type II insulin-like growth factor (IGF) receptor from rat placenta was tested for its specificity in inhibiting receptor binding of 125I-IGF II and for its ability to modulate IGF II action on rat hepatoma H-35 cells. The specific binding of 125I-IGF II to plasma membrane preparations from several rat cell types and tissues was inhibited by the anti-IGF II receptor Ig. Affinity cross-linking of 125I-IGF II to the Mr = 250,000 type II IGF receptor structure in rat liver membranes was blocked by the anti-receptor Ig, while no effect on affinity labeling of insulin receptor with 125I-insulin or IGF I receptor with 125I-IGF I or 125I-IGF II was observed. The specific inhibition of ligand binding to the IGF II receptor by anti-receptor Ig was species-specific such that mouse receptor was less potently inhibited and human receptor was unaffected. Rat hepatoma H-35 cells contain insulin and IGF II receptor, but not IGF I receptor, and respond half-maximally to insulin at 10(-10) M and to IGF II at higher concentrations with increased cell proliferation (Massague, J., Blinderman, L.A., and Czech, M.P. (1982) J. Biol. Chem. 257, 13958-13963). Addition of anti-IGF II receptor Ig to intact H-35 cells inhibited the specific binding of 125I-IGF II to the cells by 70-90%, but had no detectable effect on 125I-insulin binding. Significantly, under identical conditions anti-IGF II receptor Ig was without effect on IGF II action on DNA synthesis at both submaximal and maximal concentrations of IGF II. This finding and the higher concentrations of IGF II required for growth promotion in comparison to insulin strongly suggest that the Mr = 250,000 receptor structure for IGF II is not involved in mediating this physiological response. Rather, at least in H-35 cells, the insulin receptor appears to mediate the effects of IGF II on cell growth. Consistent with this interpretation, anti-insulin receptor Ig but not anti-IGF II receptor Ig mimicked the ability of growth factors to stimulate DNA synthesis in H-35 cells. We conclude that the IGF II receptor may not play a role in transmembrane signaling, but rather serves some other physiological function.

Insulin activates the appearance of insulin-like growth factor II receptors on the adipocyte cell surface.

To evaluate the mechanism of insulin action to increase rat 125I-labeled insulin-like growth factor II (125I-IGF-II) binding to rat adipocytes, we raised a potent rabbit antiserum against purified IGF-II receptors from rat placental membranes. The antiserum elicited a positive reaction at a 1:5000 dilution against purified IGF-II receptor in an ELISA and markedly inhibited 125I-IGF-II binding to adipocyte plasma membranes when added prior to the growth factor. Immunoprecipitation lines formed between agar plate wells containing antiserum versus IGF-II receptor, both in the presence and absence of 1 microM IGF-II, indicating that binding of anti-receptor Ig to the IGF-II receptor is not affected by occupancy of the IGF-II binding site. Intact adipocytes treated with or without insulin were incubated with anti-IGF-II receptor Ig, washed, and further incubated with 125I-labeled goat anti-rabbit IgG to monitor the amount of anti-receptor Ig bound. Insulin induced parallel increases in anti-IGF-II receptor Ig binding (2.4-fold) and 125I-IGF-II binding (3-fold) to the isolated cells. The dose-response relationship of insulin action on 125I-IGF-II binding and anti-receptor Ig binding was essentially identical with a half-maximal effect at approximately 0.07 nM insulin. That insulin does not act to expose new types of antigenic sites on IGF-II receptors was indicated by the demonstration that control adipocytes could readily adsorb the anti-receptor Ig. These data demonstrate that increased numbers of IGF-II receptors are displayed in an exposed position on the adipocyte cell surface in response to insulin.

Calcium movement and membrane potential changes in the early phase of neutrophil activation by phorbol myristate acetate: a study with ion-selective electrodes.

To quantitate calcium movements and membrane potential changes in stimulated neutrophils, we have measured net fluxes of Ca2+ and of the lipophilic cation tetraphenyl phosphonium by a very sensitive ion-selective electrode system. Activation of neutrophils by 3 X 10(-8) M phorbol 12-myristate, 13-acetate induces a release of approximately 20% of total cell calcium, with an initial lag period of less than 10 s. The Ca2+ outflux is markedly reduced in ATP-depleted cells and in the presence of a calmodulin inhibitor, thereby suggesting that it occurs by activation of the ATP-driven Ca2+ pump of the neutrophil plasmalemma. Activation of neutrophils also induces a transiently increased exchange of medium 45Ca with cell calcium, which is measurable a few seconds after cell exposure to the stimulant and peaks at approximately 40 s. Stimulation of neutrophils after attainment of steady-state accumulation of tetraphenyl phosphonium (resting potential of -67 mV) results in a marked depolarization, with a lag period of approximately 60 s. The rate and extent of depolarization are reduced by 40 and 65%, respectively, in a low Na+ medium but are not modified by an inhibitor of anion exchange across membranes. A high-K+ medium depolarizes neutrophils without either modifying their resting oxidative metabolism or impairing stimulability by the phorbol ester. Phorbol 12-myristate, which also exhibits no effect on the oxidative metabolism of neutrophils, does not induce Ca2+ extrusion and membrane potential changes. The causal relationship between Ca2+ mobilization, membrane potential changes and activation of neutrophil functions is discussed.

ATP-driven Ca2+ pump activity of macrophage and neutrophil plasma membrane.

The results of the investigations here described permit us to conclude that macrophages and neutrophils have a peripheral, outwardly directed Ca2+ extrusion system, which is very similar to the well known Ca2+ pump of the red cell, with regard to capacity and mechanism (16, 21, 29). In fact, all the three cell types have similar maximum pumping rates (about 0.1-0.2 microgram-ions Ca2+/min/ml cells) and use ATP for extruding Ca2+. Furthermore, the plasma membrane of all the three cell types catalyzes a Ca2+-dependent ATPase reaction, which is very likely the enzyme manifestation of the Ca2+ pump activity. Further investigation is needed to establish whether the peripheral Ca2+ pump system of macrophages and neutrophils is utilized to restore steady-state levels of cytosolic Ca2+ upon cell stimulation, or is somehow involved in the triggering of cell response to various stimuli. In fact impairment of the pump activity by a cell stimulant would unbalance Ca2+ passive leaks and active cation extrusion, thereby leading to higher steady-state levels of Ca2+ in the cytosol and to stimulation of Ca2+-dependent functions (1-12).

Isolation and partial characterization of the plasma membrane of purified bovine neutrophils.

1. Large amounts of granulocytes can be isolated from bovine blood by differential centrifugation and hypotonic lysis of erythrocytes, followed by separation of neutrophils and eosinophils by centrifugation through a gradient of colloidal silica. 2. Careful homogenization of the purified neutrophils and subfractionation of the postnuclear supernatant by centrifugation through a discontinuous sucrose gradient provides a membrane fraction (at the 20/32%, w/w, sucrose interface), which collects about 20--35% of the activity of plasma membrane marker enzymes. 3. Treatment of the plasma membrane fraction with 0.5 M KCl removes some protein and activity of granule enzymes, leading to an about 20--35-fold enrichment in specific activity of plasma membrane marker enzymes. In particular, there is a 25-fold enrichment in a Ca2+-dependent ATPase, whose half-maximal reaction velocity is reached at 2.2 X 10(-7) M Ca2+. 4. High-resolution sodium dodecyl sulphate/polyacrylamide gel electrophoresis reveals a complex composition of the neutrophil plasma membrane, with about 40 polypeptides stained by Coomassie blue. Ten of these peptides are more intensely stained by the dye; their apparent molecular weight ranges from 81 000 to 34 000. All these ten polypeptides but one (which is likely to be actin) are markedly enriched in the plasma membrane fraction over the original homogenate. 5. Since bovine blood can be obtained in unlimited amounts, the procedures here described can be applied to a large-scale purification of the neutrophil plasma membrane, suitable for biochemical studies.