CD4+CD25+ T-cell populations expressing CD134 and GITR are associated with disease activity in patients with Wegener's granulomatosis

Background. An increased CD4⁺ CD25⁺ T-cell population is observedin Wegener's granulomatosis (WG). This T-cell population isnot well characterized yet and their contribution to the diseasepathogenesis remains obscure. Methods. Thirty patients with WG and 18 healthy controls (HC)were included in this study. The disease activity and extensionwere measured by the Birmingham Vasculitis Activity Score (BVAS)and the Disease Extent Index (DEI). Lymphocytes from peripheralblood were analysed by FACS for the expression of CD4, CD25,CD134 and GITR. Cytokine expression in these subsets was assessedtoo. Nasal, lung and renal tissues from WG patients were immunohistochemicallystained for CD3 and CD134. Results. The percentage of CD134⁺ as well as GITR⁺ expressingCD4⁺CD25⁺ lymphocytes was increased in patients as comparedto HC (37 ± 12% versus 27 ± 8%, P = 0.005; 18± 9% versus 11 ± 6%, P = 0.003). The expressionof CD134 and GITR showed a significant correlation with diseaseactivity (r = 0.5, P = 0.009; r = 0.55, P = 0.001). Most ofthese displayed the phenotype of effector memory T-cells (94± 4% and 91 ± 6%). CD134 T-cells were found intissues affected by WG. Conclusions. CD4⁺CD25⁺ effector memory T-cells expressing CD134and GITR seem to play a role in disease mechanisms, as suggestedby their close association with disease activity and their participationin inflammatory process.     

Myoinositol incorporation into lymphocytes of chronic renal failure patients is impaired

Incorporation of myo-[2-³H]-inosital into peripheral blood mononuclearcells (PBMNC) and T-cell enriched lymphocytes was evaluatedin in-vitro experiments in chronic renal failure (CRF) patientsand healthy subjects. Incorporation of myo-[2-³H]-inosital intothe cells of CRF patients on conservative and haemodialysistreatment was found to be impaired in comparison with that observedin normal cells. Following PHA stimulation of the cells of CRFpatients myo-[2-³H]-inosital incorporation decreased even further,while it increased in normal cells. Five hour haemodialysissession significantly depressed myoinositol incorporation intoPBMNC, while its incorporation into T-cell enriched lymphocytesremained unaffected. Myoinositol incorporation into PBMNC andT-cell enriched lymphocytes was inhibited by prostaglandinsand leukotrienes and was inversely related to the extent ofpertussis toxinsensitive G protein activation. Reduced myoinositolincorporation into uraemic PHA-stimulated PBMNC may depend atleast in part on their enhanced PGE₂ and LTB₄ release accompaniedby increased intracellular cAMP production. In CRF impairedmyoinositol incorporation into immune cells may prove the disarrangementin the early events of transmembrane signal transduction, whichmay share the responsibility for the cell-mediated immune defectin these patients.     

The effect of LPS, uraemia, and haemodialysis membrane exposure on CD14 expression in mononuclear cells and its relation to apoptosis.

BACKGROUND: Both uraemia and bioincompatible haemodialysis membranes induce mononuclear cell apoptosis. Recent reports demonstrate that spontaneous apoptosis in normal monocytes is associated with the down-regulation of CD14 molecules, whereas LPS which prevents the down-regulation of CD14 favours monocyte survival. The aim of the present study was to evaluate a possible association between mononuclear cell apoptosis and low expression of CD14 molecules. This study also investigated whether LPS affects mononuclear cell CD14 expression and the apoptosis induced by uraemia and exposure to Cuprophan (CU) membrane. METHODS: The study was performed in vitro examining the effects of CU membrane and LPS on mononuclear cells from normal subjects and from end-stage renal failure patients. Cells were analysed by flow cytometry with fluorescent monoclonal antibodies to determine CD14 expression and with Annexin-V labelling to determine apoptosis. RESULTS: In mononuclear cells from uraemic patients cultured for 48 h, there was a subset of cells with low CD14 expression; this subset of cells was not observed in normal monocytes cultured for the same period of time. Cells with low CD14 expression were also observed when normal or uraemic mononuclear cells were cultured in the presence of CU membrane. Simultaneous measurement of apoptosis and CD14 expression revealed that cells with low CD14 expression underwent apoptosis. The addition of LPS to the medium markedly reduced the number of mononuclear cells with low CD14 expression and also reduced the rate of apoptosis in these cells. CONCLUSION: Our data suggest that mononuclear cell apoptosis induced by uraemia and the CU membrane is associated with low CD14 expression. Furthermore, LPS prevented the decrease in CD14 and reduced the rate of apoptosis.     

Increased serum levels of MRP-8/14 in type 1 diabetes induce an increased expression of CD11b and an enhanced adhesion of circulating monocytes to fibronectin

The recruitment of monocytes from the bloodstream is crucial in the accumulation of macrophages and dendritic cells in type 1 diabetic pancreases. Adhesion via integrins to endothelium and extracellular matrix proteins, such as fibronectin (FN), and the production of myeloid-related protein (MRP)-8, -14, and -8/14 by recently transmigrated monocytes are thought to be instrumental in such recruitment. We determined the FN-adhesive capacity and integrin expression of monocytes of type 1 and type 2 diabetic patients and related them to the subjects' serum levels of MRP-8, -14 and -8/14. Monocytes of type 1 diabetic patients displayed an increased adhesion to fibronectin in comparison with type 2 patients and healthy control subjects but had a normal expression of the FN binding integrins CD29, CD49a, CD49d, and CD49e (although CD11b and CD18 expression was increased). MRP-8/14, which was increased in the sera of type 1 diabetic patients, induced healthy donor monocytes to adhere to FN and upregulate CD11b expression in a dosage-dependent manner. The observed MRP-induced increased adhesion of monocytes to FN and upregulation of CD11b most likely contributed to a facilitated accumulation of monocytes and monocyte-derived cells at the site of inflammation, in this case the pancreatic islets.     

Uraemic toxic retention solutes depress polymorphonuclear response to phagocytosis

Previous studies from our laboratory have demonstrated thatthe activity of the hexose mono-phosphate shunt (HMS) pathwayin phagocytosisrelated respiratory burst is disturbed in end-stagerenal disease. To determine whether uraemic solute retentionis responsible for this defect the HMS-path was evaluated bymeasurements of glucose-1-C¹⁴ utilization and determinationof ¹⁴CO₂ production in polymorphonuclear cells (PMNLs), suspendedin normal plasma or uraemic biological fluids. Normal PMNLs,while suspended in normal or uraemic plasma, were stimulatedwith either latex, zymosan or Staph. aureus; CO₂ generation(measured as DPM/10³ PMNL, normal versus uraemic plasma) wasdepressed in uraemic plasma in response to latex (from 43±5to 20±3), zymosan (from 72±8 to 47±4) (P<0.01),and Staph aureus (from 73±17 to 47±8 DPM/10³ PMNL)(P<0.05). The degree of inhibition was similar for each stimulus.To characterize the substances responsible for this defect wefractionated uraemic plasma ultrafiltrate by polarity-basedsemipreparative C₁₈ reversed phase HPLC and found a decreasedresponse to Staph. aureus in the presence of fraction 2 (from102±13 to 23±10 DPM/10³ PMNL, P<0.05), andin fractions 8 and 11 (lowest value in fraction 8, 54±14DPM/10³ PMNL, P<0.05 versus control). The pattern of HPLCelution on a gradient from 100% formiate (pH 4.0) to 100% methanolindicates that there are at least two chemically distinguishablegroups of compounds, one hydrophilic (in fraction 2), and onelipophilic (in fractions 8 and 11). We conclude that uraemicbiological fluids contain factors that inhibit HMS activityrelated to phagocytosis, and that at least two groups of componentswith different characteristics are involved.     

Impaired release of interleukin-6 from human osteoblastic cells in the uraemic milieu.

BACKGROUND: Osteoblast-derived interleukin-6 (IL-6) affects bone metabolism and is linked with a number of pathological states characterized by increased bone resorption, including osteoporosis and renal osteodystrophy. To examine the possibility that uraemia directly influences the release of this cytokine in bone, we have investigated the effect of human uraemic serum on the release of IL-6 from human osteoblast-like cells. METHODS: Individual serum samples collected from healthy male volunteers or male haemodialysis patients prior to and during a dialysis treatment were assayed for IL-6, interleukin-1beta (IL-1beta) and soluble IL-6 receptor (sIL-6R) using specific enzyme-linked immunosorbent assays. MG-63 and SaOS-2 cells were cultured in media containing pooled sera from both groups and alongside matching charcoal-stripped sera. IL-6 concentrations were determined in harvested cell supernatants after 24 h. In further experiments, media containing individual sera obtained from five patients at regular intervals during their haemodialysis treatment were incubated with MG-63 cells to determine the effects of the dialysis process on IL-6 secretion. RESULTS: Haemodialysis patients had significantly higher (n = 10, P < 0.001) circulating concentrations of IL-6 (7.0 +/- 1.6 pg/ml) than normal subjects (0.4 +/- 0.1 pg/ml), but there were no significant differences in the concentrations of either IL-1beta or sIL-6R. These serum concentrations did not change significantly during 80 min of dialysis. IL-6 release by MG-63 cells incubated with charcoal-stripped serum from normal or from uraemic subjects was similar. Incubation with untreated sera from normal subjects increased IL-6 release by approximately 6-fold above the charcoal-stripped control, whereas sera from uraemic subjects increased IL-6 release by only approximately 2- to 3-fold (normal vs uraemic of 6878 +/- 595 and 2579 +/- 169 pg/ml, respectively, P < 0.001). Similar results were seen with SaOS-2 cells. Haemodialysis did not restore the capacity of uraemic serum to augment IL-6 release to the same degree as normal serum. CONCLUSIONS: These data show that the augmentation of IL-6 release from human osteoblastic cells after incubation with normal serum is greater than after uraemic serum. This may indicate the presence of an inhibitor of IL-6 release in uraemic serum that is involved in the deranged bone turnover of uraemic patients.     

2,3 Diphosphoglycerate-haemoglobin binding in uraemic patients treated with erythropoietin. A 31P-nuclear magnetic resonance study

Using ³¹P-nuclear magnetic resonance (NMR) measurements of relaxationrate for 2,3 diphosphoglycerate (DPG) phosphorus atoms, we showedpreviously that in uraemic red blood cells the DPG-haemoglobinbinding is stronger, thus stabilizing the deoxyhaemoglobin formand hence facilitating oxygen release. Here we verified if thesemodifications of spatial environment of DPG remain in uraemicpatients treated by human recombinant erythropoietin (rHuEpo).Simultaneously we measured the intraerythrocytic ATP concentration(ATP₁) and pH (pH₁) of patients. Our results show a slight decreaseon pH₁ and ATP₁ values during rHuEpo treatment. For the DPGrelaxation rates, we observed a very weak but statisticallysignificant increase 6 months after the beginning of treatment,but we cannot attribute a physiopathological significance tothese results because of the lack of accuracy of the NMR determinationof relaxation rate in red blood cells. Therefore, the DPG-haemoglobin binding is always stronger thanin normal subjects.     

The imbalance in the ratio of Th1 and Th2 helper lymphocytes in uraemia is mediated by an increased apoptosis of Th1 subset.

BACKGROUND: In uraemia there is a reduction in the total number of T lymphocytes and an imbalance in the ratio of Th1/Th2 T-helper (Th) lymphocytes. A higher rate of apoptosis in T lymphocytes has been reported in haemodialysis patients. The aims of the present study were to assess the Th1/Th2 pattern in uraemia and to evaluate whether a relative increase in Th1 apoptosis may explain the Th1/Th2 imbalance observed in uraemic patients. METHODS: Seventeen non-dialysed uraemic patients were evaluated; eight healthy volunteers served as controls. Intracellular interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) were measured by direct intracellular immunofluorescence and flow cytometry. Apoptosis was determined by flow cytometry using annexin V or TUNEL. Mechanisms of apoptosis were assessed by determination of Fas and Bcl-2 expression. RESULTS: Cell production of cytokines is significantly higher in uraemic patients than in controls. In addition, in uraemic patients only 5.1+/-2.1% of the T lymphocytes contained IFN-gamma (Th1 cells) while 61.9 +/- 14.8% contained IL-4 (Th2 cells) (P < 0.0001). The percentage of apoptosis was 29.6 +/- 6.3% and 4.7 +/- 1.6% in Th1 and Th2 lymphocytes, respectively (P < 0.001). Fas expression was higher in Th1 than in Th2 cells and the expression of Bcl-2 was lower in Th1 than in Th2 cells. The apoptosis induced by anti-Fas antibodies was similar in both types of lymphocytes. CONCLUSIONS: In uraemia there is a reduction in the proportion of Th1 lymphocytes due to a higher rate of apoptosis in this subset of lymphocytes. Th1 from uraemic patients show a higher expression of Fas and a lower expression of Bcl-2 than Th2. This makes uraemic Th1 cells more susceptible to apoptosis. The Th1/Th2 imbalance may contribute to alterations in cellular immunity observed in chronic kidney disease patients.     

Are uraemic children immunologically compromised?

BACKGROUND: Various immunological abnormalities leading to impaired immune status have been described in uraemic adults; however, few data are available for uraemic children. METHODS: In this study, peripheral blood total lymphocyte count and lymphocyte subsets (CD3+, CD4+, CD8+, CD16+, CD20+) were evaluated, skin tests with PPD and Candida antigens were performed, and serum immunoglobulin (IgG, IgA, IgM) and complement (C3, C4) levels were measured in 30 children with end-stage renal failure (10 before dialysis, 10 on continuous ambulatory peritoneal dialysis, and 10 on haemodialysis) and the results compared with those of 15 healthy controls. RESULTS: The data showed significant lymphopenia in predialysis and haemodialysis groups. No significant change was observed in the CD4+/CD8+ ratio or in the percentages of lymphocyte subsets in either group studied, while the absolute values of some lymphocyte subsets were significantly lower in all groups as compared with controls. In skin test evaluation, only the patients in the predialysis group showed a significantly decreased response to Candida antigen. The serum immunoglobulin levels were significantly decreased in the continuous ambulatory peritoneal dialysis group as compared with the control group. CONCLUSION: Our results, together with those of other paediatric studies, reported in the literature, suggest that uraemic children are not immunocompromised, though the effects of uraemia may cause some variation in their immune status.     

Comparability of Narcotrend index and bispectral index during propofol anaesthesia

Background. The dimensionless Narcotrend^TM (NCT) index (MonitorTechnik,Germany, version 4.0), from 100 (awake) to 0, is a new indexbased on electroencephalogram pattern recognition. Transferringguidelines for titrating the Bispectral Index^TM (BIS, AspectMedical Systems, USA, version XP) to the NCT index depends ontheir comparability. We compared the relationship between BISand NCT values during propofol anaesthesia. Methods. Eighteen adult patients about to have radical prostatectomywere investigated. An epidural catheter was placed in the lumbarspace and electrodes for BIS and NCT were applied as recommendedby the manufacturers. After i.v. fentanyl 0.1 mg, anaesthesiawas induced with a propofol infusion. After intubation, patientsreceived bupivacaine 0.5% 15 ml via the epidural catheter. Forty-fiveminutes after induction, the propofol concentration was increasedto substantial burst suppression pattern and then decreased.This was done twice in each patient, and BIS and Narcotrendvalues were recorded at intervals of 5 s. The efficacy of NCTand BIS in predicting consciousness vs unconsciousness was evaluatedusing the prediction probability (P_K). Results. We collected 38 629 artefact-free data pairs of BISand NCT values from the respective 5-s epochs. Because of artefacts,another 5008 epochs had been excluded from data analysis (3855epochs for the NCT index alone, 245 epochs for the BIS aloneand 908 epochs for both indices). Mean (SD) values in awakepatients were 94 (6) for Narcotrend and 91 (8) for BIS. Withloss of the eyelash reflex, both values were significantly reduced,to 72 (9) for NCT (P<0.001) and to 77 (11) for the BIS index(P<0.001). The P_K value for loss of eyelash reflex was similarfor BIS (0.95) and NCT (0.93). Decreasing BIS values coincidedwith decreasing NCT values. A sigmoid model [NCT index=52.8+26.8/(1+exp(–(BIS–78.3)/4.8))^0.4;r=0.52] described the correlation between BIS and NCT indexin a BIS range between 100 and 50. For BIS values lower than50, a second sigmoid model with a correlation of r=0.83 wasapplied [NCT index=6.6+45.3/(1+exp(–(BIS–29.8)/2.4))^0.6 r=0.83]. The relationship between burst suppression ratio(BSR) and NCT index was best described by the following sigmoidmodel: NCT index=265/(1+exp((–BSR+108)/–49); r=0.73. Conclusions. We found a sufficient correlation between BIS andNCT index, but deviations from the line of identity in someranges require attention. Therefore, a simple 1:1 transfer fromBIS to NCT values is not adequate. Our results might serve asa blueprint for the rational translation of BIS into NCT values.     

Enhanced Gastrointestinal Absorption of Aluminium in Uraemia: Time Course and Effect of Vitamin D

The present study examines the time course of aluminium absorptionin uraemic rats vs controls and investigates the effect of vitaminD. Following an oral load of 410 µmol aluminium there wasa significant increase in the urinary excretion rate of aluminiumas early as 60 min in uraemic rats. Compared with controls thisincrease was significantly greater in uraemic animals and maximumexcretion rates (77±49 vs pre-load 2±1 nmol Al/h)were achieved after 2 h. When vitamin-D-deficient rats with normal renal function werecompared with vitamin-D-replete controls, the latter excreteda significantly greater amount of the oral dose of aluminiumin their urine (727±361 vs 359±l40nmol Al/5d;P<0.02) and the post-load increase in the serum aluminiumconcentration was more pronounced in the vitamin-D-replete animals. Aluminium administered i.v. resulted in similar urinary aluminiumexcretion rates in both groups. In uraemicrats, however, regardlessof their vitamin D status, adminis tration of 1,25(OH)₂D₃ hadno effect on the amount of urinary aluminium excretion afteroral or i.v. loads. These findings suggest that although in rats with normal renalfunction aluminium absorption appears to be partly vitamin Ddependent, 1,25(OH)₂D₃ does not further augment the enhancedgastrointestinal absorption of aluminium in uraemia.     

Diminished adhesion of CD4+ T cells from dialysis patients to extracellular matrix and its components fibronectin and laminin

Background: Cell-mediated immunity is impaired in uraemia. The recognition and ensuing interactions of immune cells, such as CD4+ T lymphocytes, with adhesive glycoproteins of the extra-cellular matrix (ECM) are mediated by integrins of the {beta}1 subfamily. We have previously demonstrated that uraemic sera inhibit the proliferation and adhesion of normal CD+ T cells to ECM components. In the present study, the adhesive capacity of CD4+ T lymphocytes of dialyzed patients (both haemodialysis [HD] and continuous ambulatory peritoneal dialysis [CAPD] was evaluated. Methods: Adhesion of CD4+ T cells from dialysis patients to intact ECM and its immobilized moieties, fibronectin (FN) and laminin (LN) was measured following phorbol-12-myristate-13-acetate (PMA) stimulation. In addition, cell surface expression of {beta}1 integrins (VLA 4-6) was determined by FACScan analysis. Results: Compared to normal cells, CD4+ T cells of dialysis patients demonstrated a significantly reduced adhesion to ECM, FN and LN (27-28 vs 52-55% P <0.001). This decreased adhesive capacity was not normalized upon incubation of the cells with normal sera. Cell surface expression of {beta}1 integrins was not modified. The inhibition of cell adhesion was more pronounced in CAPD patients (23-24% vs 29-30% in HD, P <0.02). Serum albumin correlated directly with cell adhesion. Aged HD patients' T cells demonstrated increased adhesion to ECM and its ligands, whereas a reverse trend was demonstrated in the CAPD group. Conclusions: T cells of dialysis patients exhibit abnormal adhesive activity, which may be due to an acquired cellular defect induced by uraemic milieu. CAPD patients show a greater degree of adhesion impairment, possibly due to their lower concentrations of serum albumin.     

Uraemic medium causes endothelial cell dysfunction characterized by an alteration of the properties of its subendothelial matrix

Uraemic patients suffer from haemorrhagic disorders and acceleratedatherosclerosis. To evaluate the possible role of the vesselwall in these haemostatic alterations associated with uraemia,we investigated the effect of a uraemic milieu on human endothelialcell (EC) cultures and the reactivity of the extracellular matrices(ECM) generated by these cells towards platelets. EC cultureswere exposed to a pool of sera (20% in the culture medium) obtainedeither from uraemic patients or from normal donors, and thefollowing parameters were evaluated: (1) EC viability (trypanblue exclusion test); (2) von Willebrand factor (vWF) levelsin supernatants and associated with ECM; (3) the reactivityof EC and EC-derived ECM towards platelets, measured ‘exvivo’ under flow conditions (5 min, wall shear rate 800s^–1); and (4) ultrastructure of the ECM. The viabilityof EC cultured in the presence of uraemic sera was similar tocontrols. Platelet interaction with ECM generated by EC exposedto uraemic sera was significantly reduced (P<0.05). Thisdecrease was mainly related to a reduction in platelet adhesion(9.8 ± 1.9% vs 16.7±1.8% in controls, P<0.02).VWF levels in supernatants and associated with ECM were similarto controls. Ultrastructural analysis of the ECM generated byEC exposed to uraemic sera revealed a deficient matrix. An increasedremoval of EC was observed in experiments in which EC culturedin the presence of uraemic sera were perfused with citratedblood. These results indicate that a uraemic milieu induces quantitativeand qualitative changes in the vascular subendothelium, characterizedby a less intrincate network of fibrils, as well as a decreasedattachment of EC and reduced thrombogenicity to the ECM. Thesechanges may represent another mechanism which contributes tothe haemostatic dysfunction observed in uraemic patients.     

Effect of chronic uraemia on skeletal muscle metabolism in man

Fatigue and lethargy, common symptoms in uraemia, have beenattributed to many factors. To assess possible bioenergeticcontributions to this, we examined the forearm muscle of fivepatients in end-stage renal failure using ³¹P-magnetic resonancespectroscopy. There was a small increase in the ratio of intracellularinorganic phosphate to ATP in resting muscle, suggesting anincreased cytosolic phosphate concentration. During exercise,increased phosphocreatine breakdown was accompanied by rapidintracellular acidification and an increase in calculated lacticacid accumulation in the muscle of the uraemic subjects, suggestingglycolysis dominating over oxidative phosphorylation as a sourceof ATP. After exercise, the half-time of phosphocreatine (PCr)recovery was longer in the uraemic subjects, suggesting diminishedmitochondrial function. The initial rate of PCr resynthesiswas not significantly decreased, but when account was takenof the high cytosolic ADP concentration (which drives mitochondrialoxidative ATP synthesis) the calculated maximum oxidative capacitywas significantly reduced in the uraemic subjects. Thus therewas evidence of mitochondrial dysfunction in uraemia due eitherto limitation of oxygen supply, reduced mitochondrial content,or an intrinsic mitochondrial defect. This resulted in increasedphosphocreatine depletion and increased glycolytic ATP productionduring exercise and there was partial compensation of the mitochondrialabnormality by increased ADP concentration. In three of thesepatients studied after elevation of haemoglobin with erythropoeitin(from 8 to 12g/dl), initial phosphocreatine breakdown and lacticacid accumulation during exercise were normalized, while exerciseduration and calculated maximum oxidative capacity remainedsignificantly abnormal. This suggests that anaemia contributesto these metabolic abnormalities but does not fully explainthem.     

Simultaneous measurement of extracellular fluid distribution and renal function with a single injection of 99mTc DTPA

BACKGROUND.: The initial rate of renal uptake of ^99mTc DTPA is equal to theproduct of injected dose and the glomerular filtration rate(GFR):plasma volume (PV) ratio, whilst later the plasma clearancerate constant is equal to the GFR:extracellular fluid volume(ECF) ratio. Expressing GFR in terms of body fluid volumes hasseveral attractions, and techniques for measuring it in thisform may be useful. METHODS.: GFR/PV was determined from a Patlak plot in 82 studies in 47patients undergoing routine gamma-camera ^99mTc DTPA renographyand compared with GFR/ECV, determined from the terminal slopeof the plasma clearance curve. RESULTS.: GFR/PV (y) correlated significantly with GFR/ECV (x): y= 3.2.x+5.3 ml/min/l (r=0.82, n=82, P<0.001). The ratio, GFR/PV:GFR/ECVis ECV/PV, which (x) correlated significantly with a regionalestimate (y) of ECV/PV (rECV/rPV) derived from a Patlak plotapplied to a subrenal ROI: y= 0.47x+1.14 (r=0.61, n=52, P<0.001).The initial rate constant of disappearance of ^99mTc DTPA fromthe blood, determined from a ROI over the left ventricle, reflectstracer clearance from plasma to interstitial fluid plus GFR/PV.Subtraction of GFR/PV yields a rate constant which reflectsextrarenal ^99mTc DTPA clearance, Z, as a fraction of PV. A regionalestimate of ^99mTc DTPA clearance from plasma to interstitialfluid, rZ, was derived from the Patlak plot applied to the subrenalROI and expressed in relation to rPV. Z/PV (y) correlated significantlywith rZ/rPV (x): y=2.2x+0.02 min^–1 (r=0.56, n=52, P<0.001).The distribution of ECF between plasma and interstitial fluidwas related to extrarenal ^99mTc DTPA clearance. Thus rZ/rPVcorrelated significantly with ECV/PV (r=0.55, n=52, P<0.001)and Z/PV correlated significantly with rECV/rPV (r=0.6, n=52,P<0.001). There was no correlation between renal functionand extrarenal ^99mTc DTPA clearance or distribution of ECF. CONCLUSIONS.: GFR can be measured in terms of PV and ECF volume from a singleinjection of ^99mTc DTPA. ECV/PV, rECV/rPV, and variables representingthe clearance of ^99mTc DTPA across the microvasculature canbe measured at the same time.     

In-vitro effects of cyclosporin A, FK506, 6-mercaptopurine, and prednisolone on lymphokine-activated killer cells

In transplant recipients, immunosuppressive regimens are deleteriouson natural killer (NK) and lymphokine-activated killer (LAK)cells, which beyond their well-known antitumoral activity displaymany important biological functions. In order to find whichregimens could preserve NK and LAK functions we tested the influenceof CsA, FK506, 6-mercaptopurine and prednisolone on HLA-unrestrictedcytotoxicity during an in-vitro IL-2 activation. For each drug we obtained periphal blood samples from 11 healthyvolunteers. Non-adherent PBMC were incubated 2 days with eitherCsA, FK506, 6-mercaptopurine or prednisolone, whose concentrationsranged from 0 to 10 µg/ml, in order to screen infratherapeutic,therapeutic, and supratherapeutic doses. Thereafter, 100 IUof IL-2 were added for a further 3-day culture. Before and afterthe culture, we analysed (1) the cell subsets by direct immunofluorescence staining with anti-CD3/CD16/CD56 antibodies, (2) the LAKactivity with the lysis of Daudi cells, (3) the cell proliferationwith a 24-h incorporation of thymidine. Cyclosporin and FK506 did not impair the LAK cytotoxicity northe number of LAK cells, whereas both prednisolone and 6-mercaptopurinedecreased the LAK cytotoxicity, the number of CD3^– CD16⁺CD56⁺ cells, and the thymidine uptake. As a whole, the LAK cytotoxicitywas correlated with the number of CD3^– CD16⁺ CD56⁺ cellsbut not with the number of CD3^– CD16^– CD56^–cells, and it also increased with the incorporation of thymidine.This latter was correlated with the number of CD3^– CDl6⁺CD56⁺ cells, but not with the number of CD3⁺ CD16^– CD56^–cells. We conclude that in vitro the LAK activity is impaired by prednisoloneand 6-mercaptopurine, but not by CsA or FK506, and that thedeficiency of the LAK cytotoxicity seems to be related to thedecreased number of IL-2-activated NK cells.     

T-lymphocyte activation in steroid-sensitive nephrotic syndrome in childhood

We undertook a sequential study in 29 children with steroid-sensitivenephrotic syndrome (SSNS) off treatment to seek evidence forT-cell activation in relapse. T-cell subsets and activationmarkers were analysed using two-colour flow cytometry. SolubleIL2 receptor (sIL2R) was measured in serum and urine by enzyme-linkedimmunosorbent assay (ELISA). Fifteen children were examinedin remission and subsequent relapse (group A) and fourteen remainedin remission (group B). In group A the proportion of CD4⁺ cellsexpressing the activation marker CD25 (alpha-chain of the IL2receptor) increased significantly from remission to relapse:CD4⁺25⁺ cells rose from 5.6 to 7.0% of total lymphocytes, andfrom 15.8 to 19.1% of CD4⁺ lymphocytes (paired t test: P<0.0005and <0.001 respectively). No correlations were found betweenCD4⁺25⁺ cells and plasma albumin or cholesterol concentrations.SIL2R concentration in serum did not change in relapse, butincreased significantly in urine from 272 to 592 U/mg creatinine(P<0.01). No significant difference was found in remissionbetween groups A and B. We conclude that early relapse in SSNSis associated with activation of CD4⁺ (T-helper) cells whichis not secondary due to the nephrotic state itself.