Implication of gammadelta T cells in the human immune response to cytomegalovirus.

In normal individuals, gammadelta T cells account for less than 6% of total peripheral T lymphocytes and mainly express T-cell receptor (TCR) Vdelta2-Vgamma9 chains. We have previously observed a dramatic expansion of gammadelta T cells in the peripheral blood of renal allograft recipients only when they developed cytomegalovirus (CMV) infection. This increase was long lasting (more than 1 year), was associated with an activation of gammadelta T cells, and concerned only Vdelta1 or Vdelta3 T-cell subpopulations. Analysis of gammadelta TCR junctional diversity revealed that CMV infection in these patients was accompanied by (a) a marked restriction of CDR3 size distribution in Vdelta3 and, to a lesser extent, in Vdelta1 chains; and (b) a selective expansion of Vdelta1 cells bearing recurrent junctional amino acid motifs. These features are highly suggestive of an in vivo antigen-driven selection of gammadelta T-cell subsets during the course of CMV infection. Furthermore, Vdelta1 and Vdelta3 T cells from CMV-infected kidney recipients were able to proliferate in vitro in the presence of free CMV or CMV-infected fibroblast lysates but not uninfected or other herpes virus-infected fibroblast lysates. This in vitro expansion was inhibited by anti-gammadelta TCR mAb's. These findings suggest that a population of gammadelta T cells might play an important role in the immune response of immunosuppressed patients to CMV infection.

Functional quantification of cyclosporine A and FK506 in human whole blood by flow cytometry, using the green fluorescent protein as an interleukin-2 reporter gene.

The concentration of the immunosuppressive drugs cyclosporine A (CSA) and FK506 in biological fluids is routinely determined by antibody-based assays, which for several reasons do not give accurate information on the actual level of immunosuppression in the patient. To alleviate this problem, we developed a functional reporter gene assay which uses the enhancer fragment of the interleukin-2 promoter region driving the expression of the green fluorescent protein (GFP). This construct was stably transfected in the Jurkat human T lymphoblastoid cell line. Upon stimulation of the cell recipient, the GFP was produced and evaluated by flow cytometry. Immunosuppressants acting via inhibition of interleukin-2 synthesis, such as CSA or FK506, inhibited the production of GFP in a dose-dependent manner. This assay can be performed within a working day with a good reproducibility and was more sensitive than the antibody-based assays, since its detection limit was as low as 10 ng/ml for CSA and 0.5 ng/ml for FK506. We used it for the follow up of drug level present in the blood of transplanted patients, and compared the results with those obtained with the antibody-based assay routinely carried out in our hospital. The conclusions suggest that this assay is a valuable alternative to the presently available assays for the measurement of the immunosuppressive activity found in body fluids.

One-year enzyme-linked immunosorbent assay follow-up of human interleukin for Da cells/leukemia inhibitory factor in blood and urine of 22 kidney transplant recipients.

The cytokine human interleukin for Da cells/leukemia inhibitory factor (HILDA/LIF) exerts multiple biological effects in vitro. In mice, high circulating levels of HILDA/LIF induce a wide range of pathophysiological events, some of them closely involved with immunological and inflammatory responses. Using a sandwich ELISA recognizing the natural human HILDA/LIF molecule with a threshold of 50 pg/ml in urine and 150 pg/ml in plasma, we monitored the urine and plasma HILDA/LIF levels of 22 patients in their first year after a kidney transplant. HILDA/LIF urine excretion is increased during acute rejection, and infections also trigger heavy HILDA/LIF plasma concentrations or urine excretion. In addition, this study raises the question of HILDA/LIF involvement in post-kidney-transplant phenomena such as hypercalcemia, osteoporosis, or the reversal of anemia.

HILDA/LIF urinary excretion during acute kidney rejection.

Recently, a new lymphokine called HILDA (human interleukin for DA cells) has been described and cloned. This cytokine, initially described to be produced by alloreactive T lymphocyte clones grown from a rejected human kidney allograft, is identical to other factors termed D-factor, differentiation-inducing factor, differentiation inhibitory activity, hepatocyte-stimulating factor III, and leukemia inhibitory factor. HILDA/LIF induces various effects on neural, hemopoietic, embryonic cells as well as on bone remodeling and acute phase protein synthesis in hepatocyte. In this study we demonstrate the presence of HILDA/LIF in the urine but not in the serum of kidney graft recipients during acute rejection episodes, whereas this lymphokine was detectable neither in the serum nor in the urine of kidney transplanted patients with stable renal function. These data reinforce the notion of a possible role for this lymphokine in the inflammatory and/or the immune response.

Detection of single cells secreting IFN-gamma, IL-6, and IL-10 in irreversibly rejected human kidney allografts, and their modulation by IL-2 and IL-4.

The frequency of isolated low-density graft-infiltrating cells (GIC) secreting IFN-gamma, IL-6, and IL-10 was studied in 8 cases of irreversible rejection of human renal allografts, using ELISA and SPOT-forming cells (ELISPOT) assays. The GIC were mostly CD8+ T cells, although CD4+ T cells, B cells, and macrophages could also be detected. On the average, in 10(6) cells, 189 secreted IFN-gamma, 747 IL-10, and 17114 IL-6. Culture of GIC in the presence of IL-2 resulted in an increase in the frequency of IFN-gamma-producing cells (IFN-gamma-PC), in a dose-dependent manner, with an optimal 6.5-fold increase at 50 U/ml. In contrast, 25 U/ml IL-4 decreased the frequency of spontaneous and IL-2-induced IFN-gamma-PC by 71.3% and 52.8%, respectively. Furthermore, IL-4 reduced the frequency of IL-6-PC (56.8%). By this new approach to graft rejection, it becomes easier to determine cytokine profiles within an allograft and to study their modulation by different agents. It could be a useful model of study for the development of new treatment.

Tumor necrosis factor alpha in human kidney transplant rejection--analysis by in situ hybridization.

Macrophagic infiltration and necrosis of rejected kidney transplants represent two pejorative patterns. It has been assumed that the macrophagic toxicity is mediated partly by secretion of tumor necrosis factor alpha. On the other hand, TNF is also involved in many inflammatory and immunological phenomena. We thus evaluated the expression of TNF mRNA by in situ hybridization in 6 rejected kidney transplants using a radiolabeled TNF-c DNA probe. Then the synthesis of TNF alpha protein was studied by immunohistochemistry using an anti-TNF alpha antibody. In severely rejected kidney grafts, TNF mRNA is expressed in some monomorphic infiltrating cells, mostly located in the deepest part of the cortex and around the tubes. These cells do not bind other probes, such as dopa-decarboxylase DNA or preproenkephalin RNA. They are also recognized by a monoclonal antibody directed against TNF alpha. What is more, this antibody binds with some glomerular endothelial and tubular epithelial cells that do not express TNF mRNA. These cells are likely target cells for TNF. In the normal kidney, there are no cells expressing TNF-alpha mRNA.

Induction versus noninduction in renal transplant recipients with tacrolimus-based immunosuppression.

BACKGROUND: The aim of this study was to compare the efficacy and safety of induction treatment with antithymocyte globulins (ATG) followed by tacrolimus therapy with immediate tacrolimus therapy in renal transplant recipients. METHODS: This 12-month, open, prospective study was conducted in 15 centers in France and 1 center in Belgium; 309 patients were randomized to receive either induction therapy with ATG (n=151) followed by initiation of tacrolimus on day 9 or immediate tacrolimus-based triple therapy (n=158). In both study arms, the initial daily tacrolimus dose was 0.2 mg/kg. Steroid boluses were given in the first 2 days and tapered thereafter from 20 mg/day to 5 mg/day. Azathioprine was administered at 1-2 mg/kg per day. RESULTS: At month 12, biopsy-confirmed acute rejections were reported for 15.2% (induction) and 30.4% (noninduction) of patients (P=0.001). The incidence of steroid-sensitive acute rejections was 7.9% (induction) and 22.2% (noninduction)(P=0.001). Steroid-resistant acute rejections were reported for 8.6% (induction) and 8.9% (noninduction) of patients. A total of nine patients died. Patient survival and graft survival at month 12 was similar in both treatment groups (97.4% vs. 96.8% and 92.1% vs. 91.1%, respectively). Statistically significant differences in the incidence of adverse events were found for cytomegalovirus (CMV) infection (induction, 32.5% vs. noninduction, 19.0%, P=0.009), leukopenia (37.3% vs. 9.5%, P<0.001), fever (25.2% vs. 10.1%, P=0.001), herpes simplex (17.9% vs. 5.7%, P=0.001), and thrombocytopenia (11.3% vs. 3.2%, P=0.007). In the induction group, serum sickness was observed in 10.6% of patients. The incidence of new onset diabetes mellitus was 3.4% (induction) and 4.5% (noninduction). CONCLUSION: Low incidences of acute rejection were found in both treatment arms. Induction treatment with ATG has the advantage of a lower incidence of acute rejection, but it significantly increases adverse events, particularly CMV infection.

Value of captopril renal scintigraphy in hypertensive patients with renal failure.

UNLABELLED: The aims of this study were to show the value of captopril renal scintigraphy for detecting a renovascular cause in hypertensive patients with renal failure and to assess the ability to predict the beneficial effect of revascularization on renal function. METHODS: Thirty-eight patients with renal failure (mean glomerular filtration rate = 35 mL/min) underwent renal scintigraphy after injection of 99mTc-mercaptoacetyltriglycine. Baseline scintigraphy was performed, and the test was repeated 24 h later after oral administration of 50 mg captopril given 60 min before the test. RESULTS: In 5 of 6 patients with a renovascular cause for renal failure, and 2 of 3 patients with a probable arterial pathology, scintigraphy had a high probability. The result was indeterminate in the other 2 patients. In 5 of 11 patients with negative arteriography and 14 of 18 patients with probable absence of renovascular pathology, we found a low probability of functional renal artery stenosis. Six revascularization procedures were performed and were predictive of a beneficial effect in 5 patients. Time of peak activity was an effective predictor in each case. CONCLUSION: In hypertensive patients with renal failure, captopril renal scintigraphy can detect hemodynamic dysfunction downstream from a renal artery stenosis and can predict the beneficial effect of revascularization in some cases.

Low protein and low phosphorus diet in patients with chronic renal failure: influence on glucose tolerance and tissue insulin sensitivity.

Ten patients with advanced renal failure (glomerular filtration rate 25 mL/min) were treated with a low phosphorus and low protein diet supplemented with ketoacid analogues. Before starting the diet and four months afterwards, a 50 g oral glucose tolerance test with a three step euglycemic insulin clamp was carried out. A dose-response curve of total body insulin sensitivity was plotted. By the fourth month, glucose tolerance had improved with significantly lower T0, T30, and T60 insulin levels. These results are attributed to the improvement in insulin action as demonstrated by the clamp technique. The dose-response curve had a distinctly higher plateau after dietary treatment, and the tissue sensitivity index to insulin (M/l ratio) was significantly improved. It is suggested that treatment of uremic patients with a low protein diet may reduce levels of a putative insulin inhibitor.

Effect of a ketoacid diet on glucose tolerance and tissue insulin sensitivity.

Effects of a low protein (0.3 g/kg/day) diet on glucose tolerance and tissue insulin sensitivity were studied in 25 non-diabetic and eight insulin-dependent diabetic uremic patients before and three months after dietary treatment. Carbohydrates accounted for 65% of the caloric intake in the first group and 57% in the second. In the first group, a 50 g oral glucose tolerance test showed that after three months blood glucose was significantly reduced at T60 (P less than 0.05) and serum insulin at T0, T30 (P less than 0.05) and T60 (P less than 0.02). Ten patients of the first group underwent an euglycemic, hyperinsulinemic clamp study; the tissue sensitivity to insulin index of all three clamp periods improved (P less than 0.01 for the first and second, P less than 0.02 for the third). Five patients in the second group underwent a euglycemic clamp study; glucose metabolism increased with each clamp period. Concomitantly, their daily insulin requirements decreased from 37.2 +/- 3.1 to 24.8 +/- 2.7 U/day (P less than 0.05). This conspicuous improvement observed in both groups might be related to a decrease in uremic toxin(s) derived from protein intake. Beneficial results on atherosclerosis and cardiovascular pathology may occur from the reduction of hyperinsulinism.

Effect of famotidine on renal transplant patients treated with ciclosporine A.

The influence of a 7-day course of 40 mg famotidine administered orally on the pharmacokinetics of ciclosporine A at steady-state has been investigated in 10 renal transplant patients. Famotidine did not appear to significantly alter the pharmacokinetics of ciclosporine A. This might be ascribed to the limited potential of famotidine for inhibiting microsomal enzyme function. Moreover, plasma creatinine concentrations and creatinine clearance remained stable. Our results suggest that famotidine has no noticeable interaction with ciclosporine A.

Pharmacokinetics of intravenous and intraperitoneal fosfomycin in continuous ambulatory peritoneal dialysis.

Kinetics of fosfomycin were investigated in six patients undergoing continuous ambulatory peritoneal dialysis. Each subject received both an i.v. and an i.p. 1 g dose of fosfomycin with a one week washout between doses. Fosfomycin was assayed by a microbiological diffusion technique. After intravenous injection the fosfomycin serum kinetic parameters were as followed: elimination half-life (t1/2 beta) 38.4 +/- 8.7 h; volume of distribution 0.32 +/- 0.02 l/kg; total plasma clearance 7.0 +/- 1.4 ml/min and peritoneal clearance 3.2 +/- 0.2 ml/min. Dialyzate fosfomycin concentrations reached a maximum mean value of 32.2 +/- 2.8 micrograms/ml at 4 h post-injection and fosfomycin was detectable in dialyzate samples for up to 72 hours post-dosing. After intraperitoneal instillation, fosfomycin appeared in the serum rapidly and the mean peak plasma concentration was 36.2 +/- 2.8 micrograms/ml at the 4th h. The absorption rate (ka) was 0.580 +/- 0.039 h-1 and the absorption of fosfomycin from peritoneal space was 68.4 +/- 6.0%. These data suggest a bidirectional exchange through the peritoneal membrane. Intraperitoneal administration of 1 g either 48 h apart for anephric patients or 36 h apart for patients with residual renal function may achieve therapeutic serum concentrations.

Human allotransplant of a digital flexion system vascularized on the ulnar pedicle: a preliminary report and 1-year follow-up of two cases.

Two cases of a human vascularized allotransplant of a complete digital flexion system are reported with detailed descriptions of the dissection technique and postoperative treatment. Satisfactory functional results open new prospects for this type of transplantation surgery.

[Nyctohemeral variations of cyclosporine administered orally in renal transplant patients]. / Variations nycthémérales de la ciclosporine administrée par voie orale à des transplantés rénaux.

The circadian intraindividual variations of cyclosporin blood concentrations were studied in nine renal transplant patients at steady state. The cyclosporin was administrated orally twice a day (9 a.m. and 21 p.m.). Blood concentrations were analysed by H.P.L.C. AUC = area under the drug concentration versus time curve, Cmax = maximum blood concentration, Cmin = minimum blood concentration, Tmax = Time to maximum concentration. Statistical analysis (t, Wilcoxon) shows: AUC, Cmax and Tmax are not significantly modified, Cmin (21) less than Cmin (9) (p less than 0.02). This phenomenon seems to be rather a cyclosporin metabolism variation than an absorption alteration.

[Prostacyclin in the treatment of hemolytic-uremic syndrome: apropos of a case]. / Intérêt de la prostacycline dans le traitement du syndrome hémolytique et urémique: à propos d'un cas.

A 52 year-old man was hospitalised for acute renal failure with thrombocytopenia and hemolytic anemia without oliguria. A haemolytic-uremic syndrome was diagnosed and prostacyclin infusion was started. Twenty-four hours later, the renal function improved as well as thrombocytopenia and anemia. Recovery occurred after 11 days of treatment. Haemolytic-uremic syndrome treatment is not well codified: plasmaphoresis, fresh frozen plasma, transfusions showed inconstant efficiency and data about prostacycline treatment are rare and often contradictory. Multicentric studies must be started in order to determine the precise benefit of this treatment.

[Effect of verapamil on cyclosporine-induced vasoconstriction in human or murine isolated glomerules]. / Influence du vérapamil sur la vasoconstriction induite par la ciclosporine sur glomérules isolés humains et murins.

Cyclosporin is a very potent immunosuppressant, but it often produces renal disturbances which limit its clinical use. Using an image analyzer which determines the areas of isolated glomerules, we were able to demonstrate that cyclosporin in various concentrations exerts a direct vasoconstrictive effect on human and murine glomerules. We also showed that verapamil has an almost total inhibitory effect on cyclosporin-induced vasoconstriction. These findings seem to be of interest in clinical practice to reduce the nephrotoxicity of cyclosporin.

[In situ production of interferon gamma and interleukin 6 by infiltrating cells from human kidney allografts in irreversible rejection. Modulation by cytokines]. / Production in situ d'interféron gamma et d'interleukine 6 par les cellules infiltrant des allogreffes rénales humaines en rejet irréversible. Modulation par les cytokines.

Using ELISPOT assays, we detected a significant number of cells secreting interferon-gamma and interleukin 6 among 8 irreversibly rejected kidney allografts compared with normal kidneys. Interleukin 2 increases the frequency of interferon-gamma producing cells and interleukin 4 decreases the frequency of both interferon-gamma and interleukin 6 producing cells.

[Tumor necrosis factor in graft rejection. In situ hybridization study]. / Le facteur nécrosant des tumeurs dans les rejets de greffe. Etude par hybridation in situ.

The development of necrosis and macrophage infiltration increases the risk of renal graft rejection. But the macrophages secrete the alpha form of the tumour necrosing factor (TNF) which is also involved in several immunologic and inflammatory phenomena. We therefore studied the expression of the gene TNF alpha by in situ hybridization during advanced stage rejection after renal transplantation: the grafts were infiltrated with macrophage-like cells expressing the mRNA of the TNF alpha gene, particularly deep in the cortex and in the medulla. These cells then secrete the TNF alpha molecule since they are recognized by anti-TNF alpha antibodies. These antibodies also recognize certain other glomerular endothelial and tubular epithelial cells which do not express the TNF alpha gene: these cells are undoubtedly the TNF target cells. These findings confirm the synthesis of TNF alpha in advanced stage renal graft rejection.

[Surgical indications for primary vesico-ureteral reflux in adult candidates for renal transplantation]. / L'indication opératoire concernant le reflux vésico-rénal primitif de l'adulte chez un candidat à la transplantation rénale.

Eight out of thirty five candidates for renal transplantation at the Bordeaux transplantation center, between 1975 and 1983, all presenting with primary vesico-ureteric reflux, underwent nephro-ureterectomy. The remaining twenty seven were not treated surgically. The authors consider that no operation is required when the ureter is narrow and uninfected, but that dilated refluant and infected ureters should be treated by nephro-ureterectomy, at least unilaterally. In that event, the operation is performed in two stages, with the second stage being performed at the same time as the transplantation.

[Use of the receiver's own ureter, a remedial solution in lesions of the urinary tract, following renal transplantation]. / Utilisation de l'uretère propre du receveur, solution de recours dans les lésions de la voie excrétrice, après transplantation rénale.

An analysis of eleven cases in a series of 274 kidney transplantations between 1976 and 1984 suggests that pyeloureteral anastomosis with the recipient's own ureter only involves a simple operation, that the nephrectomy of the overlying kidney would not seem indispensable, that the existence of major infectious lesions is a contra-indication, and that seven of the eleven grafts were still functional with a followup of five months to seven years.