M. Falk

The kinetics of mycophenolic acid and its glucuronide metabolite in adult kidney transplant recipients

Mycophenolic acid kinetics have been reported to vary after renal transplantation, and mycophenolic acid area under the concentration–time curve (AUC) is the best predictor of suppression of graft rejection.

Advanced donor-origin melanoma in a renal transplant recipient: Immunotherapy, cure, and retransplantation

BACKGROUND A kidney transplant recipient inadvertently contracted donor-origin melanoma, which was found to be very advanced at presentation. Withdrawal of immunosuppression failed to induce rejection, and interferon-alpha was required. When florid allograft rejection was in progress, the allograft was removed, before it was recognized that the transplanted melanoma was not being simultaneously rejected. METHODS Subsequent immunotherapy was required, which largely recapitulated treatment of recognized value in autologous melanoma and included interferon-alpha, use of cultured melanoma cells as tumor vaccine, pooled allogeneic cell vaccination, and adoptive immunotherapy using lymphokine-activated killer cells. RESULTS Prolonged immunotherapy eradicated the widespread malignancy, and the patient went on to a successful second renal transplant, with follow-up of over 24 months. CONCLUSIONS This unique case demonstrates the successful cure of advanced transplanted melanoma through the use of immunotherapy, which did not require sophisticated tumor vaccine technology, and successful retransplantation.

Characterisation of γδ T cells in renal cell carcinoma patients by polymerase chain reaction analysis of T cell receptor transcripts

Abstract Renal cell carcinomas (RCC) contain tumour infiltrating lymphocytes (TIL) but these are essentially immunosuppressed in that they do not generate effective antitumour immune responses in vivo. These TIL comprise predominantly αβ T cells, although γδ T cells are also present. The repertoire of γδ T cells in RCC, however, has not been fully investigated. To identify the γδ T cell populations infiltrating RCC, this study has characterised the γδ T cell receptor (TCR) repertoire expression in these tumours and compared this to autologous normal kidney and autologous peripheral blood. A semi-quantitative reverse transcriptase/polymerase chain reaction technique was used for amplification of rearranged TCR V-C mRNA transcripts. Primers specific for the four human TCR Vγ and six Vδ subfamilies were used, each in conjunction with a primer specific for either the Cγ or Cδ region. The specificity of the PCR products was confirmed by Southern blotting and hybridisation with an internal C region probe. A densitometry score was assigned to each DNA band and the level of V gene expression was determined as a ratio of Cδ gene expression. The γδ TCR expression in each sample was determined as a ratio of Cδ: glyceraldehyde phosphate dehydrogenase densitometry score. This demonstrated that TCR Cδ gene expression was significantly higher in RCC compared to normal kidney (P<0.019), suggesting a selective infiltration of γδ T cells into the tumour. Furthermore, we observed differences in the TCR Vγ and Vδ repertoires between RCC and peripheral blood. Vγ1 expression was significantly decreased (P<0.043) whereas there was an over-representation of Vγ4 transcripts (P<0.028) in RCC compared to blood. A significant reduction in expression of both Vδ1 (P<0.028) and Vδ3 (P<0.051) was also observed within kidney tumour compared to peripheral blood. These findings show that expression of the γδ TCR repertoire in RCC differs from that in peripheral blood and normal kidney.

Apoptosis and expression of cytotoxic T lymphocyte effector molecules in renal allografts.

Cytotoxic T lymphocyte (CTL) mediated apoptosis is thought to play a major role in the rejection of renal allografts following transplantation, however, the CTL effector mechanism that is primarily responsible for immunological rejection is unknown. The two major effector pathways of CTL killing which lead to apoptosis involve the Fas/Fas ligand (Fas L) lytic pathway, and the perforin/granzyme degranulation pathway. The expression of CTL effector molecules which influence these pathways include Fas, Fas L and TiA-1 (cytotoxic granule protein). This study has investigated apoptosis by in situ terminal deoxytransferase-catalysed DNA nick end labelling (TUNEL), and the expression of CTL effector molecules by immunohistochemistry, in renal allograft biopsies obtained from patients following kidney transplantation. Renal biopsies were classified into three histological groups; acute cellular rejection, chronic rejection, or no rejection. The extent of T-cell infiltration of renal tissues was assessed by immunohistochemical staining with an anti-CD3 monoclonal antibody. Numerous TUNEL positive cells were detected in all transplant biopsies examined; these consisted mainly of renal tubular cells and infiltrating cells, with some TUNEL positive cells also detected in the glomeruli. In the case of normal kidney tissue, renal cells also stained positive for TUNEL but there was no lymphocytic infiltration. There was significantly more T-cell infiltration observed in acute rejection biopsies compared to the no rejection biopsies. In the case of Fas L expression, there was little expression in all three biopsy groups, apart from one case of chronic rejection. Conversely, although there were no significant differences in TiA-1 expression between the three biopsy groups, TiA-1 expression was more prominent in acute rejection biopsies. Furthermore, Fas expression was significantly decreased in acute rejection biopsies when compared to those of chronic and no rejection in which Fas was predominantly localized in the renal tubular cells. These results indicate that the mechanism of CTL killing leading to the rejection of renal allografts may be different in acute and chronic rejection. Moreover, our data indicate the potential for cytotoxic granule-based CTL killing in acute renal allograft rejection but not in chronic rejection.

EXPRESSION OF CYTOKINE MRNA TRANSCRIPTS IN RENAL CELL CARCINOMA

Renal cell carcinoma (RCC) is a solid tumour of the kidney and is the most common renal neoplasm. Despite the presence of tumour infiltrating lymphocytes (TIL) in RCC, these tumours continue to progress in vivo suggesting a poor host immune response to the tumour, and the suppression of TIL effector function. Cytokines are key molecules that modulate the function of T cells. The possibility is investigated that the local production of cytokines in RCC contributes to immunosuppression of TIL. The expression of pro‐inflammatory (IFN‐γ/IL‐2) and immunosuppressive (IL‐10/TGF‐β) cytokine mRNA transcripts was determined in RCC, normal kidney and peripheral blood of RCC patients using a semi‐quantitative reverse transcriptase–polymerase chain reaction (RT‐PCR) with cytokine‐specific primers. Following Southern blot hybridization of the PCR products with internal radiolabelled oligonucleotide probes, cytokine transcript levels were measured by densitometry and expressed relative to the glyceraldehyde‐3‐phosphate dehydrogenase densitometry score. With the exception of IL‐10, there were no differences in expression of cytokine mRNA transcripts between the peripheral blood of patients and normal healthy individuals. It was found that TGF‐β transcripts were well represented in normal kidney and RCC. In contrast, the expression of IFN‐γ transcripts, while low in the majority of samples, was significantly increased in RCC when compared to normal kidney (P = 0.05). The IL‐2 and IL‐10 transcripts showed a more variable expression in normal kidney and RCC, with no significant differences in expression between the sample groups. The data demonstrating pro‐inflammatory and immunosuppressive cytokine expression in RCC do not support a prominent immunosuppressive cytokine profile in these tumours.

EXPRESSION OF APOPTOTIC REGULATORY MOLECULES IN RENAL CELL CARCINOMA : ELEVATED EXPRESSION OF FAS LIGAND

Renal cell carcinoma (RCC) is the most common renal neoplasm. Despite being infiltrated by tumour infiltrating lymphocytes (TIL), these TIL are unable to control tumour growth in vivo, suggesting that the cytotoxic capacity of TIL against RCC is impaired, or that the tumour cells are resistant to killing and therefore escape detection by the immune system. It is postulated that the expression of apoptotic regulatory molecules in RCC favours tumour cell survival. The present study has therefore determined the expression of Fas (APO‐1/CD95), Fas ligand (Fas L) and bcl‐2 in these tumours. The expression of Fas, Fas L and bcl‐2 mRNA transcripts was determined in RCC, normal kidney and peripheral blood by semiquantitative reverse transcriptase polymerase chain reaction (RT‐PCR), following RNA extraction and cDNA synthesis from tissues and cell samples. Transcript levels were measured by densitometry after Southern blot hybridization of PCR products with internal radio‐labelled oligonucleotide probes; a densitometry score was assigned to each hybridizing DNA band and expressed as a ratio of the glyceraldehyde‐3‐phosphate dehydrogenase content. In peripheral blood, the expression of Fas L and bcl‐2 transcripts was similar between patients and normal healthy individuals; however, Fas transcript expression was significantly down‐regulated in the patients’ versus normal peripheral blood (P = 0.026). Most interestingly, significantly up‐regulated Fas L expression was observed in RCC compared to normal kidney (P = 0.041). In contrast, bcl‐2 transcripts were well represented in normal kidney but markedly decreased in RCC (P = 0.021). The expression of Fas transcripts in normal kidney and RCC was variable. These data demonstrate elevated expression of Fas L transcripts in RCC, but the functional relevance of this remains to be investigated.

T cell engraftment in lymphoid tissues of human peripheral blood lymphocyte reconstituted SCID mice with or without prior activation of cells

The reconstitution of severe combined immunodeficiency (SCID) mice with human PBL (Hu‐PBL‐SCID) was assessed using fresh unstimulated PBL and anti‐CD3‐stimulated PBL. Mice were reconstituted with PBL by intraperitoneal injection of 1–2.5 × 107 PBL in PBS; controls received PBS. Successful engraftment of human PBL in SCID mice was determined by measurement of human IgG in mouse sera, polymerase chain reaction (PCR) detection of human‐specific HLA‐DRβ DNA in SCID periphery, and immunohistochemical staining of mouse tissues (spleen, lymph nodes, thymus, liver and lung) with antibodies specific for human CD45 and CD3. Human IgG was detected 1 week after reconstitution in sera of all animals that received at least 1 × 107 PBL and continued to increase for 8 weeks. Human‐specific HLA‐DRβ DNA was detected in the majority of mice 3 weeks after reconstitution but not in controls. Moreover, immunohistochemical analysis of Hu‐PBL‐SCID mouse tissues revealed the presence of human CD45+ cells in all tissues examined. CD3+ T cell engraftment was observed in lymphoid tissues irrespective of whether PBL had been activated prior to transfer or not.

Use of living donor kidneys with multiple arteries

Progression to renal failure is significantly worsened by oxidative stress in chronic inflammatory kidney disease (IgA nephropathy, antiglomerular basement membrane nephritis, focal segmental glomerulosclerosis), rhabdomyolysis (myoglobinic acute renal failure), diabetic nephropathy and in poisoning by nephrotoxic compounds such as transition metals, paraquat and drugs such as cyclosporine A and cisplatin. The membrane antioxidant vitamin E (α‐tocopherol) is examined as a potential therapeutic intervention that may help to slow the rate of decline of kidney function in such conditions. An impaired plasma antioxidant defence system is characteristic of chronic renal failure and the uremic state. Vitamin E therapy is also considered as a means of correcting plasma antioxidant status and attenuating the cardiovascular disease that accompanies kidney failure.

Post renal transplant thrombotic microangiopathy: A single centre experience

SUMMARY: Thrombotic microangiopathy (TMA) is a rare complication of renal transplantation. This study is a review of our experience of seven cases of TMA occurring between 1986 and 1995, an incidence of 0.8%. One patient had a recurrence of TMA in her graft having an episode of haemolytic uraemic syndrome as the cause of her original kidney failure. the other six patients had a rejection episode either at the time of diagnosis of TMA or a mean of 12 days prior to the diagnosis of TMA. All patients were treated with plasma exchange and those on cyclosporin had their dose reduced. Two patients had graft loss while the remainder had successful outcomes with stable graft function 3–11 years post diagnosis of TMA. We conclude that, in our experience rejection played a prominent role in the disease pathogenesis and that cyclosporin could be continued successfully albeit at a reduced dosage.