Ultrastructural analysis of peripheral blood mononuclear cells in renal allograft recipients.

Renal transplanted patients receiving immunosuppressive therapy with glucocorticoids (GC) and azathioprine show in the peripheral blood numerous large mononuclear cells. The origin and the lineage of these cells was not clearly established. In the present study we investigated the surface phenotype and the ultrastructural characteristics of this subset constituted by large mononuclear cells. From peripheral blood mononuclear cells (PBL) of allograft recipients, a cell preparation exhaustively depleted of the relatively numerous monocytes was obtained. At ultrastructural examination two main cellular populations were distinguished: the predominant one (congruent to 85%) was constituted by cells with lymphoid morphology while the other (congruent to 15%) showed myeloid appearance. The large lymphoid cells, T4 or T8 positive, did not express Ia molecules on the surface and were morphologically suggestive of cells in an intermediate stage of the cell cycle between resting and activated lymphocytes. The myeloid population was constituted by promyelocytes, myelocytes and metamyelocytes. Promyelocytes and myelocytes capable of cell division are responsible for the increase of 3H-thymidine incorporation observed in transplanted patient PBL. In conclusion our data suggest that in allograft recipients the immunosuppressive therapy with GC and azathioprine may inhibit the lymphocyte blast transformation and can influence the release of immature myeloid cells responsible for the PBL increased 3H-thymidine incorporation.     

Antibodies directed against renal collecting ducts in sera of human renal allograft recipients.

Anatibodies directed against the plasma membrane of renal collecting ducts were found in the sera of two out of 101 renal allograft recipients. The sera also reacted with the epithelium of the renal pelvis, the ureter, the ductus deferens, the skin, the oesophagus, the trachea and the bile ducts of random individuals, indicating that the antibodies were directed against a widespread epithelial antigen. The antibody activity against these structures was removed by absorption of the sera with homogenized renal medullary tissue; absorption with plasma proteins, erythrocytes, spleen leucocytes or platelets did not influence the antibody titers. In the two patients with circulating antibodies, the graft function was unremarkable eight and fifteen months after transplantation, respectively. From this study we conclude that circulating epithelial antibodies directed against the collecting ducts of the graft are of little or no clinical importance.     

Active T and EA-rosette-forming cells in human cadaver renal allograft recipients.

Active T(AT) and EA-rosette-forming cells (RFC) were determined in seventeen haemodialysis patients and twenty-two renal allograft recipients to correlate these estimates with the clinical outcome of renal allografts. Either AT- or EA-RFC or both were found to be elevated during the period of rejection crisis. Assays for AT- and EA-RFC were found to be complementary suggesting that they may be detecting two different types of rejection crises. Thus, determination of AT- and EA-RFC in the peripheral blood may prove to be useful in the routine immunological monitoring of renal allograft recipients.     

Stability of peripheral blood lymphocyte count and E-rosette forming lymphocytes in healthy adults.

Highly reproducible peripheral blood lymphocyte (PBL) count and percent E-rosette forming lymphocyte (%E-RFL) assays were developed by modifying existing procedures. PBL count assay variation was reduced by using replicate electronic white blood cell (WBC) counting and 2,000 WBC differentials. The %E-RFL assay modifications reduced variation and include the use of: (a) a capillary buffy coat isolation procedure that recovered more than 85% of the PBL without the use of chemical separation media, (b) centrifugation temperatures of 20--22 degrees C, and (c) toluidine blue staining that allowed enumeration of %E-RFL and not E-rosette forming cells, by exclusion of nonlymphoid cells. Day-to-day variation of PBL count and %E-RFL was defined by making serial determinations over a period of months. The data indicate that healthy adults maintain PBL counts and %E-RFL within narrow ranges that are specific for each individual.     

Neopterin levels in long-term renal allograft recipients

In this paper we present results dealing with neopterin levels in the urine of 21 kidney grafted patients during long-term allograft survival. Data dealing with results of urinary neopterin excretion to predict acute cellular allograft rejection during the immediate post-transplant period have been reported previously. The evaluation of patients included a physical investigation and numerous biochemical parameters as described below. We found that neopterin excretion is normal or elevated, but if elevated levels remain stable the prognosis for the allograft seems to be good.     

Invasive fungal infections in renal allograft recipients

Invasive fungal infections contribute significantly to morbidity and mortality in renal allograft recipients. We identified 29 cases of invasive mycoses on histological and/or cytological examination, out of the total 1231 renal transplants performed at our centre over a period of last 15 yrs (1989-2003). A detailed clinical analysis was performed. The time interval between transplant and the occurrence of invasive fungal infection ranged from 15 days to 10.5 yrs. Candida and Aspergillus were the most frequent offenders (66%); Candida alone accounting for 45% of the cases. The most common risk factors were post transplant Cytomegalovirus infection, diabetes mellitus and episodes of acute rejection. Fine needle aspiration cytology, bronchoalveolar lavage and esophageal brush smears aided in prompt diagnosis. Disseminated infection was associated with a high mortality (80%). Management of renal transplant recipients requires identification of risk factors and early clinical suspicion of infection. The role of prophylaxis needs further evaluation.     

Long-term renal allograft recipients from South-east Asia in the pre-cyclosporin era.

The clinical outcome of long-term renal allograft recipients in the Chinese population has not been reported previously. We analysed patients from the pre-cyclosporin era who had grafts that functioned for > 10 years. Forty-five patients (31 men, 14 women; mean age 30, follow-up duration 13.3 years), representing a 10-year graft survival of 53%, were included. Thirty-six patients (80%) received living-related allografts and 9 (20%) received cadaveric or living-unrelated renal transplantation. The mean serum creatinine at last follow-up was 1.36 mg/dl (range, 0.83-4.08). Major posttransplantation complications included: hypertension in 25 (56%), infection in 16 (36%), acute rejection in 15 (33%), lipid disorder in 13 (29%), liver disease in 7 (16%), osteonecrosis in 5 (11%), malignancy in 4 (9%), coronary artery disease in 3 (7%), and diabetes mellitus in 3 (7%). Five grafts were lost: 3 to chronic rejection, and 2 to patients with stable function who died of non-renal causes. Proteinuria correlated strongly with graft function and survival, and marginally with hypertension. In hepatitis B carriers, serum alpha-feto protein is useful in the early detection of hepatocellular carcinoma. We conclude that while patients in the pre-cyclosporin era can survive with excellent graft function beyond the first decade, the risk of complications leading to significant morbidity still remains even when patients are receiving minimal doses of immunosuppression in the second decade.     

Natural killer-cell activity in cyclosporine-treated renal allograft recipients

We prospectively studied natural killer (NK)-cell activity in 16 cyclosporine-treated renal transplant recipients. NK function remained intact in the group as a whole in the initial 6 months following transplantation. The percentage of CD16-positive cells within the peripheral blood mononuclear-cell population was highly correlated with NK activity both prior to and following transplantation in the absence of rejection. During rejection, the correlation was poor. A marked increase in NK activity occurred during 9 of 12 rejection episodes; similar increases in NK activity were rarely observed in the absence of rejection. Significant infiltrates of NK cells, as determined by expression of CD16, were not demonstrated in stained biopsy specimens obtained from rejecting allografts. Pretransplant NK activity did not predict clinical outcome of the allograft. Our results indicate that NK cells are activated during allograft rejection in cyclosporinetreated patients, but their exact role in the rejection process is unknown.     

Problems associated with pregnancy in renal allograft recipients

Of 18 pregnancies in 11 renal transplant recipients, three were terminated and in the remaining 15 (in 8 women) there were 10 live births (including one set of twins), five intrauterine deaths, and one spontaneous abortion. Graft function deteriorated in six women, from obstruction of the transplanted ureter in two, recurrent glomerulonephritis in two, rejection in one, and pelvi-ureteric junction obstruction in one. Hypertension worsened or developed in all but one of the pregnancies and proteinuria appeared in eight. Of the 10 live births only one reached 38 weeks gestation (mean 35 weeks) and four neonates were small for gestational age. One infant died early from intraventricular hemorrhage and hyaline membrane disease, one fetus had hydrocephalus, and the others were normal. Factors associated with a poor fetal outcome were deterioration in graft function during pregnancy, pre-existing hypertension, or the development of hypertension before the third trimester.     

Epstein-Barr virus associated posttransplant malignant lymphoma in renal allograft recipients.

We report two cases of posttransplant malignant lymphoma(PTML) of B cell origin associated with Epstein-Barr virus(EBV) infection. They were a 52-year-old male and a 37 year-old-female, in whom intermediate-grade diffuse malignant lymphomas of large cell type developed in the submandibular area and jejunum, respectively. DNA and RNA in situ hybridization revealed the presence of EBV-specific DNA and RNA sequences in the tumor cells.     

Diminished integrin expression on granulocytes from renal allograft recipients.

Integrin (beta 2 subunit of the LEU-CAM family, CD18) expression on peripheral blood and urinary granulocytes was studied in renal allograft recipients. Circulating granulocyte CD18 expression was normal except for patients with CMV infection. In contrast, the majority of patients with urinary tract infection had low numbers of CD18+ cells collected from urine, and the same abnormality could also be observed in approx. 50% of non-infected recipients. These disturbances were associated with transplantation, as no such deficits were seen in the infected non-transplant patients or patients with glomerular diseases on immunosuppression. Short culture of granulocytes with immunosuppressants did not diminish integrin expression. Deficient granulocyte integrin expression, especially in urinary tract, may be associated with increased susceptibility to infection in renal transplant recipients, although immunosuppression alone could not be incriminated as a sole factor responsible for this deficit.     

Diagnosis of cytomegalovirus infection in cyclosporin-treated renal allograft recipients

The relative merits of antibody response and virus shedding as markers of cytomegalovirus (CMV) infection among cyclosporin-treated renal allograft recipients were analysed. CMV-specific antibody was assayed by IgG-specific radioimmunosorbent test (RIST) and by complement fixation test (CFT). CMV shedding was assayed by virus isolation and by the rapid test immediate early nuclear antigen detection (IENAD). RIST and CFT detected seroconversion in similar numbers of patients, but the former test was the more sensitive when CMV antibody was sought in pretransplant sera to differentiate primary from recurrent infection. IENAD detected or excluded CMV shedding for more urine specimens than virus isolation (462/515 [90%] vs. 366/515 [71%]), but the reverse applied to saliva specimens (33/57 [58%] vs. 54/57 [95%]). The high specificity of IENAD allowed positive results by IENAD to be accepted when virus isolation failed to provide a result. IENAD was, however, less sensitive than virus isolation even when specimens yielding CMV by IENAD, but no result by virus isolation, were included in the analysis (27/44 [61%] vs. 38/44 [86%]). Assays of both antibody response and virus shedding were required to maximise the diagnosis of recurrent CMV infections, but the former assay detected all primary CMV infections. The diagnostic implications of these results are discussed.     

Expression of FasL gene in T cells of renal allograft recipients.

FasL molecule expressed on activated T cells induces apoptosis in Fas-expressing cells. It is possible that apoptosis induced by FasL is involved in the process of allograft destruction brought about by infiltrating T cells. The aim of our study was to evaluate expression of FasL gene in peripheral blood T cells of renal allograft recipients (RAR). We have studied 25 patients: 16 with uneventful stable course (RAR-S) and nine during biopsy proven chronic rejection (RAR-CH). The relative expression of FasL mRNA compared with that of beta-actin was established by semi-quantitative RT-PCR. We have found that FasL gene expression was significantly increased in T cells of RAR-CH compared to RAR-S (P<0.01). Our results suggest that T cell expression of FasL gene is increased during chronic rejection. Therefore, this phenomenon may pay a role in allograft injury associated with that process. Further studies are needed to unravel possible clinical consequences of observed differences in T cell expression of FasL.     

Mechanisms of CML hyporesponsiveness in long-term renal allograft recipients

Long-term, HLA disparate, renal allograft recipients were assessed by a dose-dependent CML (cell-mediated lympholysis) assay for evidence of donor-specific CML hyporesponsiveness. The frequency of cytolytic T-cell (CTL) precursors capable of responding to donor alloantigen was also examined by a limiting-dilution assay and application of Poisson distribution statistics. Four recipients were found to have depressed CML responses against donor class I HLA antigens (mean 2-5% specific lysis), whereas significant responses against third party targets were noted (15-60% specific lysis). These data are consistent with an antigen-specific defect in the recipient CTL effector mechanism. To determine whether or not this may be due to a deletion of anti-donor alloreactive cells, a sensitive limiting-dilution assay was developed in conjunction with Poisson distribution statistics to obtain the frequency of both anti-donor and anti-third party CTLp present in recipient PBLs. A simultaneous reduction in the number of alloreactive clones and the presence of an active suppressor population were defined, suggesting that several mechanisms may operate concurrently in long-term renal allograft recipients with well-functioning grafts.     

Peripheral blood allogeneic microchimerism in lung and cardiac allograft recipients.

We have been investigating two parameters, donor antigen-specific hyporeactivity and peripheral blood allogeneic microchimerism, to determine whether these parameters will predict a chronic rejection-free state and which recipients may be candidates for steroid withdrawal. We have identified donor antigen-specific hyporeactivity for 33% (16/48) of lung and 23% (11/47) of heart recipients. For both organ groups, the hyporeactive subgroup experienced a lower incidence of chronic rejection. The probability of donor antigen-specific hyporeactivity predicting a chronic rejection-free state is 100% for lung and 91% for heart recipients. We have identified peripheral blood allogeneic microchimerism for 77% (20/26) of lung and 36% (9/25) of heart recipients tested at 12-18 months posttransplant. Donor antigen-specific hyporeactivity correlates with a critical level of donor cells in lung recipients; the probability of high peripheral blood allogeneic microchimerism levels predicting a chronic rejection-free state in lung recipients is 100%. The results in heart recipients are not as clear with a short-, but not long-term, trend of higher chimerism levels correlating with the development of donor antigen-specific hyporeactivity. These results illustrate the usefulness of immmune parameters to predict long-term graft outcome in an organ-specific manner.     

Plasma interleukin 2 receptor levels in renal allograft recipients

Shed/soluble interleukin 2 receptor (IL2R) was measured by an enzyme-linked immunosorbent assay (ELISA) in serial samples of plasma from 32 patients with renal allografts. Patients on chronic dialysis (pretransplant) had elevated IL2R levels which fell toward normal after transplantation. Patients with acute rejection and viral infection had significantly higher levels of plasma IL2R than did patients with stable renal function or with cyclosporine nephrotoxicity (all P less than 0.005). Acute renal failure from other causes (renal artery stenosis, hemolytic-uremic syndrome) did not have a comparable rise in IL2R. The assay of shed/soluble IL2R may have diagnostic value in the clinical management of allograft recipients, a possibility that deserves further clinical evaluation.