Eeva von Willebrand

Monitoring of Human Renal Allograft Rejection with Fine-needle Aspiration Cytology

We have performed serial fine‐needle aspiration biopsies from human renal allografts undergoing acute rejection episodes. After subtraction of blood background, the method enables a numerical evaluation of the composition of the inflammatory infiltrate. In all rejection, T lymphoblasts and especially B plasmablasts were prominent at the beginning, whereafter the blast cell response subsided if only traces of Other inflammatory cell types were seen in the aspirates, the rejections were clinically mild, usually lasting less than 5 days. If the blast response was accompanied by a considerable influx of lymphocytes but only few mononuclear phagocytes, the clinical course was intermediately intensive, although these rejections were usually reversible. If, however, the blast cells and lymphocyte were accompanied by large numbers of monocytes, and if the maturation of blood‐borne monocytes into tissue macrophages was evident, the rejections were severe and usually irreversible. Thus the appearance of large numbers of macrophages in the aspirates is a bad prognostic sign, indicating an unfavourable course and irreversible rejection.

Expression of HLA‐ABC and ‐DR Locus Antigens on Human Kidney, Endothelial, Tubular and Glomerular Cells

The distribution of the major histocompatibility complex antigens on the various cellular structures of the human kidney was analysed by using a modified Staphalococcus aureus Cowan I method Conventional alloantisera and heterologous antisera raised against Isolated molecules were used for the HLA‐ABC antisera were expressed on all types of kidney passenger leucocytes, on vascular endothelial cells, and on kidney tubular cells, but not substantially on the glomerular podocytes, The DR antigens were strongly expressed on (a fraction of) the passenger lymphocytes and on the kidney vascular endothelial cells, weakly on the passenger monocytes, but not measurably on the urine‐producing apparatus of the kidney—that is, on the glomerular and tubular cells.

In situ effector mechanisms in rat kidney allograft rejection: I. Characterization of the host cellular infiltrate in rejecting allograft parenchyma

Abstract The infiltrating inflammatory cells were recovered with collagenase and DNase from rejecting rat kidney allografts and autografts in conditions where the enzyme treatment did not affect the expression of subclass-specific surface markers. As the differential distribution of the inflammatory cells in the dispersate was similar to the distribution of inflammatory cells in tissue imprints, and as any major blood contamination was excluded, we consider the results representative of the composition of the in situ infiltrate. At the peak of rejection on Day 6 after the transplantation, approximately 30% monocytes, 17% macrophages, 31% lymphocytes, 6% (T) lymphoblasts, and 10% (B) plasmablasts and plasma cells were present in the graft. The blast cell response, pathognomonic to immune activation, was less prominent in the recipient spleen, blood, and lymph nodes. Twenty-three percent of the infiltrating lymphocytes expressed the (T-cell-specific) Pta.A.1 surface antigen(s) and 14% were surface Ig positive. The remaining lymphocytes were double-negative “null cells.” In preparative cell electrophoresis most of the allograft-infiltrating lymphocytes carried the low electrophoretic mobility, characteristic to resting B cells. Approximately 70% of allograft-infiltrating macrophages and 50% of infiltrating monocytes but only 30% of the monocytes present in the recipient spleen expressed the Fc receptor to IgG, suggesting an activation (or increase in avidity) of this receptor during the influx of mononuclear cells into the site of inflammation and during maturation of monocytes into tissue macrophages. There was a strong in situ proliferative activity, far stronger than in the central lymphatic system of the recipient rat. After 1 hr in vivo pulse labeling with [3H]thymidine 24% of the infiltrating inflammatory cells carried the label. Most of the labeled cells were blasts or lymphocytes, but a small albeit distinct number of labeled monocytes were also present in situ . In contrast to the recipient spleen, where most of the labeled lymphoid cells had a high electrophoretic mobility of resting T cells, in the infiltrate most of the labeled lymphoid cells had a slow mobility of resting B cells.

Long-term consequences of different immunosuppressive regimens for renal allografts.

The long-term effects of four different immunosuppressive regimens on renal allografts have been investigated up to four years. A total of 128 recipients of first cadaveric renal allograft were randomized, after an initial induction period, to receive either triple drug therapy—i.e., (low-dose) cyclosporine, azathioprine, and methylprednisolone, or any possible combination of two drugs—i.e., Aza plus CsA, Aza plus MP, and CsA plus MP. The actual four-year graft survival rates for the triple therapy, Aza plus CsA, Aza plus MP, and CsA plus MP groups were 72%, 69%, 75%, and 59%, and patient survival rates were 78%, 81%, 81%, and 84%, respectively, with no significant differences. The cumulative number of chronic rejections up to 4 years was 0.09, 0.29, 0.25, and 0.34 per patient per group (P=ns), respectively. At 2, 3, and 4 years posttransplantation, the graft function was significantly worse in the Aza plus MP group compared with the triple therapy group (JP<.05). Of the 98 patients who did not have type I or II diabetes at the time of transplantation, 17 developed posttransplantation diabetes mellitus or an abnormal glucose tolerance test within 2 years posttransplanta-tion. All these patients had received steroids and none of the patients without steroids had these abnormalities. At two years the mean cholesterol level was highest in the Aza plus MP group, 6.8 mmol/L and lowest in the group receiving triple therapy, 5.8 mmol/L (P=ns). The use of (low-dose) CsA was not associated with lipid abnormalities or with disturbances in glucose metabolism. A protocol graft biopsy was performed at two years on all functioning kidneys, and the histological changes were scored blindly. No CsA-specific changes, except isometric vacuolation in tubuli, were found. Histological alterations characteristic of chronic rejection were expressed as “chronic allograft damage index.” Chronic allograft damage index was lowest in the triple therapy group, 1.5, compared with the other groups, 3.2–4.3 (P=.01), indicating the least histopathological change in the triple therapy group. In conclusion, this long-term study did not show any serious cyclosporine-related side-effects when used in low dose in combination with two other drugs. Some side-effects, such as posttrans-plant diabetes mellitus and probably some lipid abnormalities, could rather be traced to a higher dose of steroids. Moreover, the triple drug therapy was more efficacious than any double drug regimen in the prevention of chronic histological changes in renal allografts.

Lymphoid cell subclasses in rejecting renal allograft in the rat

We have quantitated the frequency of lymphoid cell subsets in rejecting renal allografts and in the spleen of the allograft recipient during drug-unmodified rejection in the rat. The number of inflammatory (white) cells in the graft was approximately similar to the number of white cells responding to the allograft in the recipient spleen. The inflammatory population of the graft consisted of lymphoid cells and mononuclear phagocytes, with increasing numbers of macrophages toward the end of rejection. Analysis of allograft cellular dispersates with monoclonal antibodies directed to the lymphoid cell subsets demonstrated that although the majority of allograft-infiltrating lymphocytes were T cells, a sizable B-cell proliferation and immunoglobulin synthesis was associated with the inflammatory response of rejection. Within the T-cell subset, the T suppressor/killer cells predominated in the graft whereas the predominant lymphoid cell subset responding to the allograft in the recipient spleen was the T helper cell.

In situ effector mechanisms in rat kidney allograft rejection. III. Kinetics of the inflammatory response and generation of donor-directed killer cells.

We have isolated and identified the infiltrating inflammatory cells from rejecting rat kidney allografts. The first host cells lo appear in the graft, already a few hours after the transplantation are monocytes and lymphocytes. Both T and B lymphocytes contribute to the infiltrate: at early stages of rejection most of the infiltrating lymphocytes have the high electrophoretic mobility of (resting) T cells, whereas later during the rejection most of the infiltrating lymphocytes display the slow mobility of (resting) B cells. The blast response follows 2 days after the influx of lymphocytes. The (B) plasmablast response lakes place somewhat earlier and is higher in magnitude than the (T) lymphoblast response. Macrophages appear 1.5 days after the influx of monocytes. The inflammatory cells proliferate rapidly: after 1 h of pulse‐labelling with 3H‐TdR in vivo up to 24% of the infiltrating leucocytes are labelled. Most labelled cells are blast cells or lymphocytes. although a small but distinct population of labelled monocytes is also detected in situ. The in situ blast and proliferative responses precede the corresponding responses in the host central lymphatic system, i.e. spleen, blood and lymph nodes. The inflammatory leucocytes arc isolated from the allograft parenchymal cells via I e velocity sedimentation. They are strongly and specifically cytotoxic in the 6 h 51Cr release assay to donor‐derived lymphoid target cells in vitro. The peak in situ cytotoxic activity in the graft lakes plate already on day 5 after the transplantation, whereas in the central lymphatic system the cytotoxic cells are detected later and peak values are obtained only after the activity In situ has declined. The findings emphasize the role of the graft as the site of sensitization of kidney transplantation (peripheral sensitization) and the complex nature of the inflammatory response responsible for allograft rejection.

Fine-needle aspiration biopsy in the monitoring of liver allografts. II. Applications to human liver allografts.

Serial fine-needle aspiration biopsies (FNAB) were used for clinical monitoring of human liver allografts. Nine liver allograft recipients were monitored with FNAB at 1–3-day intervals. No complications were recorded. All patients underwent at least 1 inflammatory episode of acute rejection; altogether 11 episodes, all reversible, were recorded. The inflammatory infiltrate consisted mainly of lymphoid cells, including lymphoid blasts, with minor involvement of monocytes, monoblasts, and macrophages. Further analysis of lymphoid cell subpopulations by immunoperoxidase techniques demonstrated an increase of T cells during rejection, both the CD4 (T4) and CDS (T8) subsets were increased. A slight increase of B cells in the graft was also seen. The CD4/CD8 (T4/T8) ratio was first low, peaked at the onset, and decreased toward the end of the episode. No clear correlations to the intragraft cellular events were recorded in corresponding blood specimens. However, an episode of eosinophilia was seen in the blood at the beginning of rejection, correlating with fever in the recipient. Degenerative changes in the parenchymal cells and bile droplets in the hepatocytes, indicating cholestasis and hepatocyte damage, were seen during all episodes of rejection, and these changes persisted even 10 days after the inflammation had subsided. The FNAB-findings correlated well with biochemical laboratory parameters, but the diagnosis of rejection could be established by the FNAB already 1–5 days earlier than elevated serum values indicated liver dysfunction.

Fine-needle aspiration cytology of human renal transplants

Abstract To establish diagnostic cytological criteria for acute allograft rejection, fine-needle aspiration (FNA) biopsies were performed on human renal allografts undergoing acute rejection and on allografts with no clinical evidence of rejection. There were no complications related to the procedure. Cytological evaluation of the aspirate and the blood samples taken concurrently were done from May-Grunwald-Giemsa (MGG)-stained cytocentrifuge preparations. The cytological characteristics of the various parenchymal and inflammatory cells are described in detail. The increment value, i.e., percentage of inflammatory cells in the infiltrate minus the percentage of white cells in the blood, gives a representative picture of the in situ inflammation. In nonrejecting allografts only a few monocytes and lymphocytes were seen in the FNA. Upon onset of rejection lymphocytes and T and B blast cells appeared in the aspirates, and the number of monocytes was increased. In cases of reversible rejection, all inflammatory cell elements disappeared concurrently with the regeneration of the graft function. In cases of irreversible rejection, the blast cells disappeared from the aspirates or decreased in number, but the number of monocytes increased and increasing numbers of monocytes matured into macrophages.

OKT48 ratio in the blood and in the graft during episodes of human renal allograft rejection

Abstract We have analyzed the frequency of T helper (Th) and T suppressor/killer ( Ts k ) lymphocytes in the blood and in the renal allograft during episodes of rejection and during quiescence. Monoclonal OKT4 and OKT8 antibodies were used to mark the Th and Ts k cells, respectively. Density centrifugation-separated mononuclear leukocytes and FACS IV cell sorter or the Staphylococcus aureus rosette assay were used to determine the ratio in the blood, with concordant results. Fine needle aspiration biopsy (FNAB) and the Staph. assay were used to demonstrate the lymphocyte subtypes in the graft. The mean OKT 4 8 ratio in the blood was significantly lower in the transplant recipients than in healthy controls (1.1 ± 0.7 vs 1.8 ± 0.2, respectively, P = 0.000). The individual variation was, however, high and no correlation between the OKT 4 8 ratio in the blood and the inflammatory episodes in situ was observed. During 19 of the 25 episodes of inflammation, the dominant lymphocyte subtype in the graft was the Ts k cell. In the remaining six cases it was the Th cell. All rejection episodes of the former type were reversible, in the latter type, four out of six were irreversible.

Leishmaniasis Diagnosed from Bronchoalveolar Lavage

A 51-year-old renal transplant patient, whose spleen had been removed 11 years ago, was admitted to hospital for elective surgery, which was cancelled as she developed spiking fever and nonproductive cough and her general condition deteriorated. After 2 weeks, leishmaniasis was unexpectedly diagnosed from a bronchoalveolar lavage specimen, which had been subjected to parasitological examination under the suspicion of pneumocystosis. Isoenzyme typing identified the parasite as Leishmania infantum. The patient had visited Malaga, Spain, twice a year, the last trip taking place 1 month before admission. Specific treatment was followed by rapid recovery without relapse during 1.5 years. Splenectomy and immunosuppressive medication obscured the clinical suspicion of leishmaniasis. The case is a reminder of the interstitial pneumonitis in leishmaniasis and emphasizes the value of broad-spectrum methods detecting a variety of parasites.