Measurement of intercompartmental fluid shifts during haemodialysis in children.

Seven children (age range 12-19 years, post-dialysis weights 23-43 kg) were studied during 20 haemodialysis sessions. Impedance between wrist and ankle (on the non-fistula side) was recorded using the Xitron 4000B analyser. A 2 ml sample of blood was taken for total protein and haematocrit from the arterial line at the start of dialysis. At approximately 20 minute intervals during dialysis, the time and volume of ultrafiltrate removed were recorded, and a simultaneous measurement of whole body impedance made over 25 logarithmically spaced frequencies in the range 5-500 kHz. A 2 ml sample of blood was also taken, from which serum protein and haematocrit were calculated. Hypotensive episodes occurred during four haemodialysis sessions. The percentage change in extracellular fluid (ECF) volume was calculated, at each sample time for each session, using the impedance measurements and ultrafiltration measurements (denoted delta Vi and delta U respectively). Changes in the intravascular volume were estimated using measurements of haematocrit and serum protein (and denoted delta Vh and delta Vp respectively). Least-squares regression gave delta Vi = 3.77 delta Vh, 1.33 delta Vp and 0.39 delta U, and r2 = 0.72, 0.94 and 0.95 respectively (p < 0.0001 in each case) for the 16 dialysis sessions without hypotensive episodes. Similar analysis of four dialysis sessions with hypotensive episodes gave similar relationships with correlation coefficients 0.64, 0.92 and 0.94. These relationships may not be accounted for by the anthropometric terms alone in the impedance equations. Impedance measurements also detected the addition of 300 ml isotonic saline given at the onset of each of the four hypovolaemic episodes. The regression equations support the following hypothesis: during haemodialysis, ultrafiltrate is removed from the intravascular volume but is replenished by fluid from the interstitial volume. The reduction in ECF volume measured by impedance (where the ECF comprises the intravascular and interstitial volumes) delta Vi is therefore greater than delta Vh and delta Vp, which only measure intravascular volume, but less than delta U since the ECF is replenished by fluid from the interstitial space. That delta Vh is greater than delta Vp may be due to protein loss during dialysis. The results suggest that whole body impedance measurements reflect changing body water distribution during dialysis in children.

The immunomodulatory compound 2-acetyl-4-tetrahydroxybutyl imidazole causes sequestration of lymphocytes in non-lymphoid tissues.

2-Acetyl-4(5)-(1,2,3,4-tetrahydroxybutyl) imidazole (THI) is an immunomodulatory compound which causes a reversible lymphopenia in mice by an unknown mechanism. In this study, we investigated the whereabouts of cells lost from the blood and the spleen during THI treatment Homing studies following is injection of fluorescently labelled splenocytes into THI-pretreated recipients showed that THI increased labelled cells in the liver, lungs and kidneys of THI-treated mice. Furthermore, the sequestration in the liver occurred just 1.5 h after injection of labelled cells with the increase still being present at 24 h after injection. Microscopic examination of liver sections indicated that fluorescent lymphocytes were clustered within the liver sinusoids in THI-treated mice, possibly associated with endothelial cells. The liver retention of lymphocytes was confirmed by immunohistochemical studies which showed a significant increase of T cells in the liver of THI-treated mice. To determine the subset of lymphocytes which are lost from the spleen and sequestered in non-lymphoid organs, lymphocytes remaining in the spleen after THI treatment were characterized. Our results confirmed that THI reduced B cells, CD4+ and CD8+ T cells and cells expressing CD62L, CD44 and IL-2R in the spleen.

Total body water measurement in renal insufficiency.

Total body water was measured in 15 children with renal insufficiency (glomerular filtration rate < 25 ml/min per 1.73 m2) using deuterium oxide dilution. Total body water was also measured using bioelectrical impedance and skinfold anthropometry in the same 15 children. There was a linear correlation (r = 0.98, P < 0.01) between total body water measured by deuterium and height2/impedance. The 95% confidence limits for estimates of total body water were -1.5 to 0.9 for impedance and 0.65-3.16 l for skinfold anthropometry when compared with deuterium dilution. Bioelectrical impedance estimation of total body water is better than the current existing non-invasive method of skinfold anthropometry.

The immunosuppressive compound 2-acetyl-4-tetrahydroxybutyl imidazole inhibits the allogeneic mixed lymphocyte reaction by sequestration of a recirculating subpopulation of T cells.

2-acetyl-4(5)-(1,2,3,4-tetrahydroxybutyl)imidazole (THI) is an immunosuppressive component of caramel food colouring that causes lymphopenia in mice and rats by an unknown mechanism. In this study we investigated some of the affects of THI on the murine immune system. Initially we showed that splenic T lymphocytes from mice treated with 50 mg/l THI in their drinking water were unable to launch a mixed lymphocyte reaction (MLR) against allogeneic stimulator cells, and had decreased and delayed interleukin-2 (IL-2) production. However, these T cells exhibited a normal proliferative response to concanavalin A (Con A), immobilized anti-CD3 monoclonal antibody (mAb) and anti-CD3 plus anti-CD28 mAb. Furthermore, the MLR response could be restored by the addition of IL-2 to the MLR culture. Homing studies using intravenous injection of fluorescence-labelled splenocytes showed that THI treatment decreased absolute numbers of labelled T and B lymphocytes in the blood and the spleen. Furthermore, these labelled cells reappeared in the blood and the spleen when mice were taken off THI, indicating that lymphocyte recirculation and splenic homing were modified reversibly by THI treatment. Cessation of THI treatment also resulted in a rapid reappearance of MLR responsiveness in the spleen, indicating that THI treatment does not functionally impair recirculating T cells. Collectively these data are compatible with the concept that a rapidly recirculating population of T cells, which produce IL-2 in an allogeneic MLR, are lost from the blood and spleen following THI treatment, and are sequestered in other, yet to be identified, tissues.

Combined treatment with enuresis alarm and desmopressin for nocturnal enuresis.

Seventy-one children with nocturnal enuresis were enrolled in a controlled trial. The children were allocated to two matched groups. Children in both groups used an enuresis alarm until the end of treatment. Children in the first group were treated with 40 micrograms of intranasal desmopressin (Desmospray) for up to 6 weeks at the start of treatment with the alarm. During the observation period treatment there were 2.3 dry nights per week in both groups. At the end of treatment there was a significant difference in the mean number of dry nights per week between the two groups (6.3 in the alarm and desmopressin group and 4.8 in the alarm group) and also in the number of children becoming reliably dry. The combination of desmopressin and alarm was particularly helpful for children with severe wetting and those with family and behavioural problems.

Acute renal failure due to xanthine stones.

A 9-month-old child with the skeletal abnormalities of Fuhrmann's syndrome presented with acute renal failure secondary to bilateral renal calculi. Hereditary xanthinuria was shown to be the underlying metabolic defect. Treatment with allopurinol was unsuccessful at reducing the xanthine excretion.

Assessment of the sensitivity of bioimpedance to volume changes in body water.

The sensitivity of whole-body electrical impedance measurements to changes in the volume of total body water in 12 children undergoing haemodialysis has been assessed. The impedance (I) of each child was measured at 20-min intervals during dialysis using the standard four-electrode technique to apply a constant current (800 microA, 50 kHz) between the wrist and ankle on the non-fistula side of the patient. The ultrafiltration volume (U) was also recorded. A simple electrical model suggests that U = aHt2 ((1/I0)-(1/I)), where I0 is the whole body impedance at the start of dialysis, a is a constant and Ht is patient height. No significant changes in I were measured on 4 patients undergoing dialysis without ultrafiltration, whereas in 8 patients undergoing ultrafiltration and dialysis I increased. Linear regression analysis and the above equation gave a mean value for a = 0.566 1 Ohm/cm2 (coefficient of variation = 3%), (mean r = 0.97), values comparable to those values obtained from isotope dilution studies. Predicted fluid loss in 8 patients following a single dialysis session gave a mean overestimate of 4.3% (limits of agreement 27.3% and -19.7%), although in 6 of the patients agreement was to within 6%. Changes in impedance reflect changes in total body water in children undergoing haemodialysis and are relatively insensitive to factors such as the possible differences in electrolyte levels between these patients.

Volumetric control of continuous haemodialysis in multiple organ failure.

A system for precise volumetric control of continuous haemodialysis and its use in providing renal replacement treatment in the intensive care unit to 10 children with multiple organ failure are described. The system, termed slow efficient dialysis, provided effective clearance of urea, creatinine, potassium, and phosphate. It provided precise control of the volume of ultrafiltrate removed in a prospective manner ('dial up' fluid balance) to reduce haemodynamic instability and fluid management problems. The ease of use of this system for intensive care nurses meant that the system ran without the assistance of a second intensive care or renal nurse.

Reliability of haemodialysis urea kinetic modelling in children.

The reliability of urea kinetic modelling (UKM) in paediatric haemodialysis was tested by comparing results of the classic variable volume model (UKM3), a recently introduced two-sample modification of this (UKM2) and direct quantification by a partial dialysate collection method (PDC). Urea generation rate (G) was also found from a 1-week collection of dialysate and urine (OWC). Nine children aged 2-18 years and weighing 10.6-39.9 kg were examined over 1 week (25 treatments). UKM3 and UKM2 gave almost identical results, but deviated from PDC and OWC. The two indirect methods overestimated G by 24% and 18%. However, the correlations between the results were very high for all variables and all methods (r > or = 0.96). Repeating UKM3 and UKM2 mid-week for 5 consecutive weeks, the following coefficients of variation were found: for the normalised whole body urea clearance (Kt/V) 10% and 11%, respectively; for normalised protein catabolic rate 17% and 14%. It is concluded that all tested methods can be used, but each method requires its own reference interval. Results of UKM seem to vary somewhat more than in adults. This should be considered when assessing children by such methods.

Effect of lipoarabinomannan and mycobacteria on tumour necrosis factor production by different populations of murine macrophages.

Tumour necrosis factor (TNF) production is an important pathological mediator in mycobacterial infections, and yet little is known of the factors which influence its production. We have studied the influence of murine macrophage heterogeneity and activation state on TNF production following mycobacterial stimulation in vitro. Lipoarabinomannan (LAM) from strains of Mycobacterium tuberculosis and Myco. avium differentially stimulated TNF production in thioglycollate-elicited macrophages in a dose-dependent manner. In comparison, resident peritoneal macrophages produced much less TNF when stimulated with LAM, dead mycobacteria or lipopolysaccharide (LPS). In contrast, zymosan stimulated resident macrophages to a higher degree than thioglycollate-elicited cells. Another comparison between bone marrow and thioglycollate-elicited macrophages showed that both responded to LPS, but only the latter was stimulated significantly by H37Rv LAM. This may indicate that LAM stimulation of macrophages takes place through a different pathway than both zymosan- and LPS-stimulated TNF production. Also, in vitro activation of peritoneal macrophages with interferon-gamma (IFN-gamma), increased TNF response to several stimuli. Our studies indicate that the pathology of mycobacterial infections through TNF production may be influenced by the type and activation state of the macrophage which responds to that infection.

Characterization of lymphocyte receptors for glycosaminoglycans.

This paper describes attempts to isolate and characterize glycosaminoglycan (GAG)-binding molecules on the surface of lymphocytes and lymphoma cell lines and relate their expression to splenic and lymph node homing capacity. Initial binding studies with radiolabelled GAG and rosetting studies with GAG-coupled erythrocytes revealed that there are receptors on lymphocytes for the major classes of GAG (i.e. hyaluronic acid, chrondroitin sulfates, heparin), but lymphocytes bind heparin much more avidly than other GAG species. Analysis of the binding of solubilized radiolabelled cell-surface molecules to immobilized GAG revealed cell-type specific expression of GAG-binding molecules. Thus, each of four lymphoma cell lines tested gave a characteristic pattern of GAG-binding molecules, some molecules being unique to a particular cell line and others being shared by some of the lines. Similarly, splenocytes expressed at least 10 distinct GAG-binding molecules with molecular weights (MW) ranging from 10,000 to 100,000, whereas thymocytes expressed additional GAG-binding proteins of 190,000 and 250,000 MW. Furthermore, splenocytes differed from thymocytes by possessing a unique family of cell-surface molecules which reacted with each GAG. Immunoprecipitation studies demonstrated that the GAG-binding molecules on splenocytes did not correspond to any of the cell-surface antigens tested, notably the cell adhesion molecules MEL-14, CD11/CD18 and CD44, although CD8 bound weakly to heparin. Four lymphoma cell lines with well-characterized migration properties were examined for GAG-binding molecules which may control lymphocyte migration. It was found that no one GAG-binding protein could be correlated with the entry of cells into a particular lymphoid organ. Nevertheless, the role of GAG-binding molecules in the subsequent positioning of lymphocytes within lymphoid organs requires further investigation.

Receptors on lymphocytes for endogenous splenic glycosaminoglycans.

Previous studies have shown that lymphocytes carry cell surface receptors for sulphated polysaccharides (SPS), and SPS recognition may play a role in lymphocyte migration and positioning in vivo. This paper describes attempts to isolate and characterize the endogenous glycosaminoglycans (GAGs) of murine spleen and determine whether splenic lymphocytes carry cell surface receptors for these GAGs. A procedure was devised for isolating GAGs from murine spleen in good yield and high purity and the GAG preparation was then radiolabelled for subsequent binding studies. It was found that the splenic GAGs bound to murine splenocytes in a saturable, rapid and reversible manner with only a small subpopulation of the splenic GAG preparation being involved in binding. This reactive species was chondroitinase ABC-resistant and nitrous acid-sensitive, indicative of a heparan sulphate/heparin-like molecule. Furthermore, using immunofluorescent flow cytometry studies it was demonstrated that the majority of spleen cells have receptors for these GAGs. Subsequent ion-exchange fractionation and SDS-PAGE analysis of chondroitinase ABC-resistant GAGs confirmed that the splenic GAG recognized by splenocytes was a heparan sulphate/heparin molecule of approximately 20,000 MW with a binding affinity to splenocytes of approximately 5 X 10(-8) M. Additional binding inhibition studies indicated two possible binding sites for splenic GAGs on the splenocyte surface, one being fully inhibited by a range of SPS such as heparin (both coagulant and anticoagulant forms), pentosan sulphate, fucoidan, dextran sulphate, lambda- and iota-carrageenan, and the second being partially inhibited by kappa-carrageenan. The possible relevance of these heparan sulphate/heparin receptors on splenocytes to lymphocyte positioning in vivo is discussed.