Well-functioning Grafts Research Articles

Neutrophil β2-Microglobulin and lactoferrin content in renal failure patients

Multiple dysfunctions of polymorphonuclear leukocytes (PMNLs) contribute significantly to the increased morbidity and mortality among patients with end-stage renal disease. In the present study, we measured the PMNL content of β2 -microglobulin (β2 m) and lactoferrin in different states of renal insufficiency and after kidney transplantation. PMNLs were lysed ultrasonically and, after centrifugation, both proteins were assayed in the supernatant by enzyme-linked immunosorbent assay technique. Despite marked differences in plasma β2 m levels, no significant difference in PMNL content of β2 m and lactoferrin could be shown among the groups analyzed. There was also no correlation between plasma β2 m level and PMNL β2 m content. In control subjects, as well as in renal allograft recipients with a well-functioning graft, PMNL β2 m level correlated positively with PMNL lactoferrin level (pooled data, r = 0.55; P < 0.001; n = 55). Both proteins are considered to colocalize in peroxidase-negative PMNL granules. However, no correlation was found in the azotemic and uremic patient groups. Standard immunofluorescence staining of control PMNLs showed a cytoplasmic granular distribution of both granule proteins. However, in PMNLs of uremic patients, lactoferrin shifted to a perinuclear localization. PMNLs obtained from uremic individuals failed to elicit an increase in lactoferrin release after stimulation with the chemotactic peptide f-Met-Leu-Phe compared with PMNLs obtained from healthy volunteers. These data indicate abnormalities in uremic patients of PMNL granule lactoferrin content and release that are reversible after successful renal transplantation.

Effect of increased arterial resistance index on long-term outcome of well-functioning kidney grafts

Effect of increased arterial resistance index on long-term outcome of well-functioning kidney grafts

Extracellular matrix proteins in organ transplantation.

Extracellular matrix proteins in organ transplantation.

Cyclosporine-associated thrombotic microangiopathy in renal allografts

The association between cyclosporine (CsA) and thrombotic microangiopathy (TMA) in renal allografts is well documented. However, predisposing factors and therapy guidelines are not adequately characterized. We reviewed 188 patients with kidney or kidney-pancreas transplants who were treated between January 1994 and December 1996 with prednisone, CsA, or tacrolimus, and azathioprine or mycophenolate. We analyzed 50 patients who had graft biopsies: 26 with TMA and 24 with no TMA, as well as 19 patients with well-functioning grafts who never required biopsy. TMA was observed in 26 of 188 renal graft recipients (14%). TMA was confined to the allograft kidney without any systemic evidence in 24 of the 26 patients. At the time of the diagnosis of TMA, 24 of the patients were on CsA, with 19 on the microemulsion form. Conversely, 5 of 18 control patients with no graft dysfunction were on the microemulsion form of CsA (P = 0.0026). Graft loss was seen in 8 of 26 patients with TMA. Conversion from CsA to tacrolimus resulted in a one-year salvage of graft function in 13 of 16 (81%) patients. TMA was the cause of renal graft dysfunction in 14% of renal graft recipients and was associated with the use of the microemulsion form of CsA. Systemic signs of TMA were rare, underscoring the importance of the graft biopsy in making the diagnosis. The most successful strategy was switching from CsA to tacrolimus, with good graft function in 81% of the recipients one year after the TMA episode.

Stabilization of Bone Mass After Renal Transplant With Preemptive Care

OSTEODYSTROPHY is a well-known complication of renal insufficiency and failure. The loss of nephron mass and the resultant decreased excretory function leads to an increased retention of toxic metabolites, hyperphosphatemia, and metabolic acidosis. In addition, the loss of nephron mass leads to decreased renal synthesis of 1,25dihydroxy vitamin D3, which causes decreased calcium absorption from the gastrointestinal tract. While transplantation corrects many of these metabolic complications leading to osteodystrophy, the addition of corticosteroids, and preexisting hyperparathyroidism may nullify the benefits of a well-functioning graft on bone metabolism. A prospective approach was undertaken at our institution to provide therapy aimed at decreasing the rate of bone loss after transplantation. Preemptive therapy included: goal of tapering steroids to less than 10 mg per day by the second year post-transplant; routine calcium and vitamin D supplementation; surgical parathyroidectomy when indicated; and hormone replacement in post-menopausal females. Routine bone densitometry by dual energy x-ray absorptiometry (DEXA) and markers of bone metabolism were measured at 1, 6, 12, and 24 months after renal transplant.

CYCLOSPORINE LEVEL CHANGES IN PEDIATRIC RENAL TRANSPLANT (TX) PATIENTS DURING GROWTH HORMONE THERAPY.

57 Pediatric renal Tx patients with persistent growth failure may be treated with Growth hormone (GH). However, GH therapy has been associated with increases in serum creatinine levels and possible rejection. Recent studies in humans have revealed an increase in cytochrome P450 activity (CYP3A4 hepatic enzyme) with GH treatment. However, there have been no studies to examine the effect of GH treatment on Cyclosporine (CSA) metabolism. Therefore, 15 patients (12 boys and 3 girls) with 11 cadaveric and 4 living-related well-functioning grafts (ages from 5-14 yrs), who received GH therapy at least 1 year after renal Tx, were studied retrospectively. Measurements of whole blood 12 hour trough CSA levels by the TDx monoclonal assay were examined at the time of GH therapy initiation and at 3, 6, 9, and 12 months pre and post-GH therapy. The initial average CSA dose was 6.1±3.1 mg/kg/day; the average prednisone dose was 0.12±0.04 mg/kg/day. Other immunosuppressive therapy included lmuran (Azathioprine) at 1.2±0.2 mg/kg/day or Cellcept (Mycophenolate mofetil) at 35.3±12.1 mg/kg/day. GH therapy was discontinued in one patient after 4 months of treatment because of creatinine elevation and acute rejection, confirmed by renal biopsy. In another patient, GH treatment was discontinued after 11 months for acute rejection, also confirmed by renal biopsy. Of the remaining 13 patients, 9 patients completed 12 months of GH treatment, 3 patients completed 9 months and 1 patient completed 6 months of treatment. CSA dose adjustment to maintain appropriate immunosuppression was done when indicated. A significant decrease in CSA level (p< 0.05) was noticed at 3 and 12 months post-GH treatment compared with baseline, in the absence of a significant change in the weight-adjusted CSA dose in mg/kg/day. Paired Two-tailed Student t test was used (Table).Using ANOVA (single factor), CSA levels at 3, 6, 9 and 12 months during GH therapy were significantly lower than baseline, 3, 6, 9 and 12 months prior to GH therapy (p=0.0145), at a time when no difference in CSA dose was detected. These results suggest that GH treatment decreases CSA levels. Increasing CSA dosage may be needed during GH treatment to prevent acute or subclinical rejection.

Infection-associated macrophage activation accelerates chronic renal allograft rejection in rats.

The influence of infection-associated cellular activation on chronic rejection of kidney grafts was assessed in an established rat model by administration of lipopolysaccharide (LPS), an endotoxin and a potent stimulator of various cell populations including mononuclear cells and renal epithelial cells. Lewis recipients of F344 kidneys were treated with low-dose cyclosporine (1.5 mg/kg/day x 10 days). Animals with well-functioning grafts received a single dose of LPS (2 mg in 1 ml of NaCl, intraperitoneally) at 4 or 8 weeks after engraftment. Untreated control rats, which later experienced chronic rejection, were given 1 ml of NaCl. Administration of LPS during the early quiescent phase of chronic rejection accelerated the chronic process, functionally (proteinuria), morphologically, immunohistologically, and by reverse-transcriptase polymerase chain reaction as compared with untreated controls. Infiltration of macrophages and their associated factors was especially affected. As the later events of chronic rejection seem to be mediated primarily by macrophages and their products, administration of LPS accelerated the tempo and activity of these cells in the development of chronic rejection. These findings may explain the clinical observation that infection may be an important risk factor for chronic allograft rejection.

Anti-CD4 monoclonal antibody-induced allograft tolerance in rats despite persistence of donor-reactive T cells.

Although CD4-targeted therapy abrogates acute rejection and may induce permanent graft acceptance in rodents, little is known about the mechanisms of long-term graft survival in these models. Recently, we have shown that treatment with a nondepleting anti-CD4 monoclonal antibody (mAb) (RIB-5/2) induces long-term survival of renal, heart, and skin allografts in strong major histocompatibility complex I/II incompatible rat strains. Here, we demonstrate that the development of major histocompatibility complex-specific and tissue-nonspecific tolerance rather than graft adaptation is responsible for long-term anti-CD4 mAb-induced transplant survival. Donor-specific but not third-party heart and pancreatic islet grafts were accepted permanently without adjunctive therapy in long-term kidney allograft recipients, and infusion of naive or alloimmune splenocytes failed to break the tolerant state. Interestingly, alloreactive T cells were not depleted in these long-term survivors, as ex vivo donor-specific mixed lymphocyte reaction was largely unaffected. The reverse transcriptase-polymerase chain reaction analyses of long-term renal allografts before and after donor-specific antigen challenge revealed no changes in CD3 mRNA level, but showed up-regulation of CD25, interleukin (IL) 2, interferon (IFN) gamma, IL-4, and IL-10 mRNA in the early phase, suggesting the presence of alloreactive T cells in tolerant rats. At later time points, the expression of IFN-gamma declined rapidly, whereas IL-4 persisted, resulting in a reversal of IFN-gamma/IL-4 ratio. Our data demonstrate the stability of anti-CD4 mAb-induced tolerance despite persistence of alloreactive T cells, suggesting the role of active tolerance-maintaining mechanisms. The T helper (Th) 1/Th2 shift may be involved in this regulatory process, as anti-CD4 mAb prevents acute graft-deteriorating rejection by effectively blocking Th1 responses, and well-functioning grafts may tolerize themselves by inducing regulatory cells.

Single-center experience with renal transplantation in patients with Wegener's granulomatosis.

Between 1980 and 1995, 13 patients with end-stage renal disease due to Wegener's granulomatosis received 14 renal transplants (10 cadaveric, 4 living related). The mean follow-up in the 13 successfully transplanted patients was 50 months (4-107 months). One patient had primary nonfunction and received another graft 4 months later. Three episodes of acute rejection occurred in two patients, and one of these patients lost her graft due to severe vascular rejection 4 months after transplantation. Two patients died with well-functioning grafts (one of metastatic cancer and one of sepsis). One patient presented with perisinusitis and had a mild recurrence of Wegener's disease. None of the patients developed recurrent disease in the transplanted organ. At the last follow-up, the mean creatinine (+/-SD) in the 12 patients with functioning grafts was 1.6 +/- 0.6 mgdl. We conclude that renal transplantation is an excellent treatment for renal failure due to Wegener's granulomatosis. Recurrence of the disease is uncommon in patients under immunosuppression, but careful monitoring is extremely important.

Higher renal functional reserve in well-functioning renal grafts

Higher renal functional reserve in well-functioning renal grafts

Periodic assessment of indirect allorecognition pathways in renal transplant recipients with long-term well-functioning graft

Periodic assessment of indirect allorecognition pathways in renal transplant recipients with long-term well-functioning graft

IMMUNOLOGICAL MONITORING IN TRANSPLANTATION

Sixty eight patients who had undergone live related donor renal transplantation (LRD), were evaluated for soluble interleukin-2 receptors (sIL-2R), tumour necrosis factor alpha (TNF-α) and autoantibodies against IgG(Fab')2 and IgG(Fc), at pre- and various post-transplant intervals. Serum sIL-2R levels were significantly elevated in hemodialysed patients awaiting transplantation (mean 259.2 ± 90.5 pmol/L) as compared to healthy volunteers (mean 52.6 ± 16.7 pmol/L). In 96 samples obtained from patients with well-functioning grafts (WFG), the post-transplant sIL-2R levels (135.6 ± 65.4 pmol/L) were significantly lower (p < 0.001) than their pretransplant values. Eight patients with cyclosporin-A (CsA) nephrotoxicity, 14 with reversible acute tubular necrosis (ATN) and 4 patients with partial surgical obstruction, revealed moderate levels (99.0 ± 13.7, 184.1 ± 47.5, 156.7 ± 40.4 pmol/L respectively). On the other hand, 29 patients with acute rejection episodes, 11 with chronic rejection and 8 with infections had significantly higher levels (307.9 ± 89.3, 253.3 ± 68.6,345 ± 110.6 pmol/L), (p < 0.001). TNF-α levels were also raised in rejection and infective episodes but were not statistically significant. Serum anti-IgG(Fab')2 levels were found higher (0.407 OD) in WFG as compared to those with declining graft functions (0.279 OD). On the contrary high pre- and post-transplant anti-IgG(Fc) activity was associated with increased graft rejection and lower survival rate.

Plasma Carnitine Kinetics during Orthotopic Liver Transplantation

Background: Carnitine is synthesized mainly in the liver and plays an essential role in the transport of fatty acids in liver mitochondria for subsequent oxidation and energy production. Methods: The plasma concentrations of free carnitine, acylcarnitine, total ketone bodies, lactate, pyruvate, and hepatocyte growth factor (HGF) were measured during liver transplantation. Results: The plasma free carnitine and acylcarnitine concentrations and the lactate to pyruvate ratio in patients with compromised grafts (group A) were significantly higher than those in patients with well-functioning grafts (group B) after reperfusion. The acylcarnitine concentration in group B decreased after incision, but it remained at a high level in group A. Significant correlations were found between the concentrations of HGF and free and acylcarnitine after reperfusion. Conclusion. The accelerated flux of carnitine in the graft may be associated with deterioration of energy metabolism in the graft. An increased acylcarnitine concentration may reflect impaired liver regeneration.

Detection of donor-specific hyporesponsiveness following late failure of human renal allografts

Limiting dilution assays to measure the frequency of interleukin-2-secreting peripheral blood T cells were carried out in patients, whose renal allografts had failed due to acute rejection (9 patients) and in patients whose grafts failed more than two years after transplantation without any recent evidence of acute rejection. Using a modified form of the assay we demonstrate that nearly half of 18 patients whose renal transplants had failed after more than two years have low or undetectable HTLp frequencies against donor, but not third-party DR antigens. No such difference was observed in any of the nine patients studied whose transplants were lost from early acute rejection. These results provide the first indication that, as in rodent models of transplantation, T cell unresponsiveness towards donor MHC antigens can occur following prolonged residence of an allograft in humans. Furthermore, the results suggest that chronic rejection may be driven by mechanisms other than direct allorecognition. The assay may be a valuable tool to study the evolution of donor-specific direct T cell alloresponsiveness in patients with well-functioning grafts.

Retroperitoneal placement of living related adult renal grafts in children less than 5 years of age - a feasible technique?

Abstract The extraperitoneal approach was used for transplantation of adult renal grafts in eight children under 5 years, three of them weighing less than 10 kg. Usually, children of this size are approached trans-peritoneally. The modified procedure included partial mobilization of the right liver to provide enough space for retroperitoneal graft placement and positioning of the vascular anastomoses, invariably to the aorta and caval vein. With this regimen, no significant space problems were encountered in recipients as small as 9kg, and particularly, neither arterial nor venous complications occurred. Currently, at a mean follow-up of 2.4 years, all patients are alive with well-functioning grafts. Thus, the extraperitoneal approach represents a feasible and successful procedure in the pediatric living related renal transplantation setting for recipients under 10 kg of weight.

Complete withdrawal of immunosuppression in allograft recipients. A study in rhesus monkeys.

The influence of pretransplant blood transfusions on kidney allograft survival after cessation of immunosuppressive treatment was studied in 11 rhesus monkeys. The animals were conditioned by three pretransplant blood transfusions. After an induction treatment with cyclosporine (CsA), the immunosuppression was stopped and the natural course of the graft was followed. In two monkeys long-term graft survival without immunosuppression was obtained (2.5 and 4.25 years). In a third monkey, permanent allograft acceptance was achieved after complete cessation of immunosuppression. The monkey is still alive with a well-functioning graft for more than 13 years after cessation of immunosuppression. This monkey had received CsA for 12 months, two MHC DR-matched blood transfusions, and no repeated mismatches between graft and blood transfusion donors. We speculate that blood transfusions may influence allograft function in two opposite ways. As reported previously in man, MHC class-II-matched transfusions appear to beneficially influence allograft survival. This effect seems to be negated by a mismatched MHC class II antigen in the blood transfusion donor which is also present in the organ donor -- a so-called repeated mismatch. Further studies in rhesus monkeys are required to confirm and extend these results. In the future, these observations might help in developing a protocol that opens up the possibility of cessation of immunosuppression in transplant patients.

Clinical significance of serum hepatocyte growth factor in orthotopic liver transplantation

Hepatocyte growth factor (HGF) plays a key role in the regulation of liver regeneration after hepatocyte damage. Changes in HGF production reflect the status of the regeneration process. Serum concentrations of HGF and energy substrates were measured during and after liver transplantation in 30 recipients. In the patients with compromised grafts (group A) HGF concentrations were persistently high after reperfusion, whereas in the patients with well-functioning grafts (group B), HGF concentrations decreased rapidly and remained low 4 hours after reperfusion. The patients in group A who died had persistently high concentrations of HGF. The surviving patients with reversible primary graft dysfunction in group A exhibited low concentrations 48 hours after reperfusion. The decrease in HGF concentration preceded the decrease in aspartate aminotransferase concentration. The metabolic parameters that reflect carbohydrate metabolism by the graft paralleled the changes in HGF. HGF may be more sensitive and specific in predicting early graft function than prothrombin time, ratio, aspartate aminotransferase, or arterial ketone body ratio. The determination of HGF levels after liver transplantation may yield valuable information for evaluating early graft function and making an early decision to repeat a graft procedure in an acutely ill patient.

The effects of nondepleting CD4 targeted therapy in presensitized rat recipients of cardiac allografts.

The immunosuppressive effects of RIB-5/2, a nondepleting anti-rat CD4 monoclonal antibody (mAb), were analyzed in a well-defined model of accelerated cardiac allograft rejection. (LEW x BN)F1 hearts are rejected within 24 hours in LEW hosts presensitized with BN skin grafts at day -7. Treatment with RIB-5/2 mAb (3.5 mg/day i.v.) at days -7 and -1, prolonged cardiac allograft survival to the median of >62 days. The long-term recipients rejected acutely third-party (Wistar-Furth) test skin grafts, without an adverse effect on the survival of the original cardiac transplants. Lymphocytes harvested from mAb-treated hosts significantly decreased proliferative responses of donor cells in mixed leukocyte reaction. The cell activation and cytokine elaboration patterns were evaluated at the mRNA and protein levels by competitive template reverse transcriptase polymerase chain reaction and immunohistochemistry, respectively. Cardiac allografts in CD4 mAb-treated rats at 24 hours displayed reduced CD3, CD25, tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-2, interferon (IFN)-gamma, and IL-10 mRNA levels as compared to those in rejecting grafts. Equal amounts of IL-4 mRNA were detected throughout in both animal groups; the expression of IL-10 mRNA increased progressively in the treated hosts. In contrast, IFN-gamma was consistently depressed after mAb therapy. The mRNA levels coding for CD3, CD25, tumor necrosis factor-alpha, IL-1-beta, and IL-2 genes were comparable in long-surviving and rejecting allografts. The staining for IL-2R, IL-2, and IFN-gamma was diminished, whereas the staining for IL-4 was either unaffected or enhanced in well-functioning grafts in RIB-5/2 mAb-treated hosts. The untreated recipients elicited strong circulating IgM allo-Ab response, which peaked around the time of cardiac rejection and then switched to IgG allo-Ab 4-7 days after heart transplantation. Treatment with RIB-5/2 mAb decreased IgM and prevented the switch into the IgG allo-Ab response. In conclusion, the ability of RIB-5/2 mAb treatment to combat accelerated rejection and to produce long-term graft acceptance is unprecedented in our experience in this model. These data provide new insights into the complexities of the cellular and humoral responsiveness, contributing to the the induction of donor-specific unresponsiveness in sensitized hosts. This study, along with our previous reports, indicate that an immune deviation in which intragraft Th1-type cytokines (primarily IFN-gamma) are diminished and Th2-type cytokines (IL-4 and IL-10) are maintained represents the common effector mechanism of CD4 mAb regimens in recipients of vascularized organ allografts.

Retroperitoneal placement of living related adult renal grafts in children less than 5 years of age--a feasible technique?

The extraperitoneal approach was used for transplantation of adult renal grafts in eight children under 5 years, three of them weighing less than 10 kg. Usually, children of this size are approached transperitoneally. The modified procedure included partial mobilization of the right liver to provide enough space for retroperitoneal graft placement and positioning of the vascular anastomoses, invariably to the aorta and caval vein. With this regimen, no significant space problems were encountered in recipients as small as 9 kg, and particularly, neither arterial nor venous complications occurred. Currently, at a mean follow-up of 2.4 years, all patients are alive with well-functioning grafts. Thus, the extraperitoneal approach represents a feasible and successful procedure in the pediatric living related renal transplantation setting for recipients under 10 kg of weight.

Downregulation of intragraft IFN-gamma expression correlates with increased IgG1 alloantibody response following intrathymic immunomodulation of sensitized rat recipients.

A single intrathymic injection of donor-specific spleen cells (2 x 10(7)) abrogates accelerated (24 hr) rejection of LBNF1 cardiac allografts in presensitized LEW rats and prolongs graft survival to about 11 days. This effect is donor-specific, gamma-irradiation-sensitive, thymus-dependent, and requires no concomitant therapy. We have recently shown that following intrathymic alloantigen administration, there is an earlier and increased systemic production of alloreactive IgM, and subsequently a premature isotype switching to IgG with the predominant IgG1 and IgG2a alloantibody responses. There is also a preferential binding of these IgG subclasses to the endothelium of well-functioning allografts. In this work, we analyzed the early cell activation and related cytokine elaboration patterns at the mRNA and protein levels by competitive template RT-PCR and immunohistochemistry, respectively. We found that prolonged cardiac allograft survival following intrathymic administration of donor spleen cells in presensitized rats was associated with markedly depressed intragraft IFN gamma mRNA and protein expression. Moreover, intrathymic allostimulation has led to a defect in the IL-2 pathway as the expression of IL-2 and IL-2R protein at the graft site was inhibited despite stable IL-2 mRNA levels. The inhibition of cell activation was also demonstrated by reduced MHC class II and the lack of ICAM-1, and TNF-alpha expression by immunohistochemistry. The expression of biologically active IL-12 (p70) by mononuclear and endothelial cells was detected in rejecting grafts between 3 and 12 hr. The well-functioning grafts after intrathymic allostimulation were devoid of IL-12 (p70), which in turn may have contributed to the downregulation of IFN-gamma mRNA and protein. Treatment with r.IFN-gamma, but not with r.IL-2, recreated the rejection response, and the characteristic IgG subclass pattern associated with accelerated graft loss. Hence, intrathymic immunomodulation with alloantigen results in selective inhibition of IFN-gamma-producing cells and a preferential upregulation of IgG1 alloantibodies. These data support the notion of the interlocked immunoregulatory roles of cytokine and alloantibody networks in rat allograft recipients.