The bridge between transplantation and regenerative medicine: Beginning a new Banff classification of tissue engineering pathology.

The science of regenerative medicine is arguably older than transplantation-the first major textbook was published in 1901-and a major regenerative medicine meeting took place in 1988, three years before the first Banff transplant pathology meeting. However, the subject of regenerative medicine/tissue engineering pathology has never received focused attention. Defining and classifying tissue engineering pathology is long overdue. In the next decades, the field of transplantation will enlarge at least tenfold, through a hybrid of tissue engineering combined with existing approaches to lessening the organ shortage. Gradually, transplantation pathologists will become tissue-(re-) engineering pathologists with enhanced skill sets to address concerns involving the use of bioengineered organs. We outline ways of categorizing abnormalities in tissue-engineered organs through traditional light microscopy or other modalities including biomarkers. We propose creating a new Banff classification of tissue engineering pathology to standardize and assess de novo bioengineered solid organs transplantable success in vivo. We recommend constructing a framework for a classification of tissue engineering pathology now with interdisciplinary consensus discussions to further develop and finalize the classification at future Banff Transplant Pathology meetings, in collaboration with the human cell atlas project. A possible nosology of pathologic abnormalities in tissue-engineered organs is suggested.

Donor horseshoe kidneys for transplantation.

BACKGROUND: Experience with donor horseshoe kidneys for transplantation is very limited. Currently, horseshoe kidneys may be underutilized for transplantation because of the greater incidence of vascular anomalies, associated renal anomalies, and predisposition to renal disease. METHODS: In this report, we review five transplantations using horseshoe kidneys: the largest reported institutional experience. In addition, a review of all published cases in the English literature is performed. RESULTS: All five patients underwent successful renal transplantations with a median follow-up of 35 months. One patient lost his kidney from recurrent disease soon after transplantation. CONCLUSION: With appropriate reconstruction of the vessels, careful division of the isthmus, and avoidance of ureteral obstruction, long-term data revealed good graft survival of donor horseshoe kidneys in renal transplantation.

Measurement of mycophenolate mofetil effect in transplant recipients.

BACKGROUND: Immunosuppression involves the nature of the immunosuppressive agents and individual differences in patient factors. We investigated whether the effect of mycophenolate mofetil (MMF) is measurable using an in vitro measure of immunocompetence and related its effects to cyclosporine (CsA) in vitro. METHODS: Liver or kidney transplant recipients receiving prednisone; CsA or tacrolimus; and MMF, azathioprine (AZA), or neither, were studied. Immunocompetence was assessed by one-way mixed lymphocyte culture using patients' peripheral blood leukocytes (PBL) and three validated stimulators. The effect of immunosuppressive agents added in vitro on normal PBL stimulation by Staphylococcus enterotoxin B was determined by the carboxyfluorescein diacetate succinimidyl ester measurement of division. RESULTS: Patients receiving MMF had an average immunocompetence level of 12+/-23, compared with 39.7+/-65 and 25.5+/-42 for those receiving AZA or neither AZA nor MMF, respectively. Thus, there was an approximately 80% suppression of the response in the MMF group. Assessment of normal cell division revealed that CsA allowed multiple cell generations but suppressed the numbers of cells in each, whereas MMF blocked proliferation into subsequent generations. Addition of clinically relevant levels of mycophenolic acid, the active agent for MMF, added to more moderate levels of CsA, was required to achieve greater than 80% suppression, consistent with the degree of immunocompetence depression measured in patients. CONCLUSIONS: These data provide the novel finding that the effect of MMF treatment on patients is measurable in their PBL as decreased immunocompetence in vitro. The effect of MMF on normal PBL approximates the 80% inhibition that we found in patients. Moreover, the effect of MMF on cell division provides a rationale for the superior effectiveness of regimens including MMF.

Human monocytic ehrlichiosis: an emerging pathogen in transplantation.

BACKGROUND: The spectrum of disease caused by Ehrlichia spp. ranges from asymptomatic to fatal. Awareness and early diagnosis of the infection is paramount because appropriate therapy leads to rapid defervescence and cure. If left untreated, particularly in immunosuppressed patients, ehrlichioses may result in multi-system organ failure and death. METHODS: We report the second case of human monocytic ehrlichiosis (HME) in a liver transplant recipient, and review the literature. RESULTS: The patient presented with fever and headache, had negative cultures, and despite broad-spectrum antimicrobial coverage appeared progressively septic. After eliciting a history of tick exposure we treated the patient empirically with doxycycline. The diagnosis of HME was confirmed by 1) polymerase chain reaction (PCR) for Ehrlichia chaffeensis, 2) acute and convalescent serum titers, and 3) in vitro cultivation of E chaffeensis from peripheral blood. CONCLUSION: Although human ehrlichioses are relatively uncommon, they are emerging as clinically significant arthropod-borne infections. Although epidemiological exposure is responsible for infection, immunosuppression makes patients more likely to succumb to disease. A high index of suspicion and early treatment results in a favorable outcome.

Decreased effect of immunosuppression on immunocompetence in African--Americans after kidney and liver transplantation.

Several studies indicate that the poorer outcomes for African--Americans after transplantation may be due to decreased effectiveness of immunosuppressive agents. Using an in vitro test of immunocompetence (IMC), we measured the effects of immunosuppression on African-American, compared with Caucasian, kidney or liver transplantation recipients. The IMC result was the highest of three mixed lymphocyte culture responses using validated stimulator cell pools. A total of 293 tests were done in Caucasians and 144 in African--Americans. Overall, the IMC for African--Americans was 38, compared with 19 for Caucasians (p<0.01). This decreased effect of immunosuppression (higher IMC) was the same for liver as for kidney transplant recipients, occurred at the 2--3-yr interval, and was largely in patients of tacrolimus (FK506), with a strong but not significant trend in cyclosporine (CYA) recipients. The two groups were on the same nominal immunosuppression and FK506 and CYA levels were not different. We conclude that African-Americans retain more immune responsiveness on equivalent dose immunosuppression, notable particularly in years 2--3 after transplantation when earlier graft loss occurs in this group.

Positive effect on T-cell regulatory apoptosis by mycophenolate mofetil.

The regulatory benefit of apoptosis (activation-induced cell death, AICD) in T cells may be impacted by immunosuppressive agents. We examined this for mycophenolate mofetil (MMF) compared with cyclosporine (CYA). Peripheral blood leukocytes (PBL) were stimulated by either Staph enterotoxin B (SEB) or by anti-CD3 plus anti-CD28. Cell division analysis (sequential reduction in carboxyflourescein diacetate succinimidyl ester, CFSE) was used to measure proliferation and determine status of different cell generations. Apoptosis was measured by annexin V staining, and FasL expression by anti-FasL antibody staining, of activated cells using flow cytometry. CSA and mycophenolic acid (MPA, the active agent of MMF) were added in titration in 3-day cultures. We found that CSA caused diminution in apoptosis but MPA increased it with SEB stimulation. The CSA effect on apoptosis was present when a more calcineurin-dependent stimulus. anti-CD3+ anti-CD28, was used but the MPA effect was less, producing a decrease only in the undivided cells. To look more directly at the differential effect on calcineurin-dependent AICD gene induction of the two agents, we measured Fas-L expression with anti-CD-3 + CD28 stimulation, and confirmed that CYA caused a major decrement in appearance of Fas-L, whereas MPA caused a converse accumulation of it. This seems to be explained by the block more distal in cell activation, resulting in a build-up of a precursor in the activation pathways. We conclude that MMF treatment may be rationale as an adjunct to calcineurin inhibitor treatment because of its converse effect on T cell regulatory apoptosis.

Shorter waiting times for hepatitis C virus seropositive recipients of cadaveric renal allografts from hepatitis C virus seropositive donors.

INTRODUCTION: The purposes of this study were: 1) to analyze the early results of cadaveric renal transplantation from either hepatitis C virus seropositive (HCV+ ) or hepatitis C virus seronegative (HCV-) donors into HCV + recipients; and 2) to determine whether HCV+ patients with end-stage renal disease (ESRD) might benefit from receiving renal allografts from HCV + donors. METHODS: From January 1997 to June 1999, 28 patients with ESRD and HCV infection underwent 29 cadaveric renal transplants. The data were reviewed retrospectively. Nineteen of the renal transplants were performed with allografts obtained from 15 HCV + donors and 10 with allografts obtained from 10 HCV- donors. The median follow-up was 16.2 months, with an average of 15.4+/-2 months. RESULTS: Recipients of HCV + renal allografts had shorter waiting times for transplantation. On average, patients who received a kidney from HCV + donors were transplanted 9+/-3 months after being placed on the transplant list, compared to 29+/-3 months for patients who received a kidney from a HCV- donor. Shorter waiting times were noted in every blood type group. There were no significant differences in rejection episodes, infectious complications, renal function, liver function, graft survival, or patient survival. CONCLUSIONS: The use of renal allografts from HCV + donors for HCV + recipients shortens the waiting time for these patients, with no short-term differences in renal and liver function, graft loss, or patient survival.

Plasmapheresis: an effective therapy for primary allograft nonfunction after liver transplantation.

BACKGROUND: The role of plasmapheresis in liver failure and hepatic coma remains controversial. Also, its use as a salvage strategy for patients with severe allograft dysfunction after liver transplantation has not been defined. This report reviews the use of plasmapheresis in primary hepatic allograft nonfunction (PNF). METHODS: From May of 1997 to October of 1998, five patients underwent plasmapheresis for PNF after other causes of immediate allograft dysfunction were excluded. These patients underwent two to five plasmapheresis procedures during which one plasma volume was removed and replaced with fresh frozen plasma (FFP) or with 50% FFP and 50% albumin. RESULTS: All recipients who underwent plasmapheresis had restoration of liver function. There was one death from pulmonary embolism, for an overall survival rate of 80%. The four surviving patients all had functioning allografts 1 year after liver transplantation. In contrast, during the same period, there were two patients in whom PNF was treated by retransplantation, and both died within 3 months after surgery with functioning allografts. CONCLUSIONS: Plasmapheresis provides an effective treatment option for PNF immediately after liver transplantation and may obviate the need for retransplantation.

Effects of glucocorticoids and cyclosporine on IL-2 and I kappa B alpha mRNA expression in human peripheral blood mononuclear cells.

To evaluate molecular mechanisms that might account for the heterogeneity in the in vitro responsiveness of individual subjects' peripheral blood mononuclear cells (PBMC) to immunosuppressive drugs, the authors quantitated in normal human cells the suppressive effects of the glucocorticoids prednisolone and methylprednisolone and of cyclosporine on interleukin-2 (IL-2) mRNA expression and IL-2 production, as well as the stimulatory effect of these drugs on IkappaBalpha mRNA expression. As expected, cyclosporine was significantly more suppressive than either glucocorticoid of IL-2 mRNA expression and IL-2 production by mitogen-stimulated PBMC, with variable degrees of inhibition in cells from individual subjects. The authors confirmed in human PBMC the stimulation of IkappaBalphamRNA expression by the glucocorticoid reported by others in HeLa and transfected Jurkat cell lines. In addition, the authors observed a stimulatory effect on IkappaBalpha mRNA expression by cyclosporine as well in 8 of 10 PBMC preparations studied, suggesting a possible role of calcineurin in the regulation of IkappaBalpha production. Interindividual variability in the intracellular mechanisms of action, possibly based on molecular polymorphisms, might be one factor contributing to differences among patients in their clinical responses to treatment with such drugs.

Lymphocyte suppression by glucocorticoids with cyclosporine, tacrolimus, pentoxifylline, and mycophenolic acid.

Methylprednisolone has been found to be significantly more suppressive than prednisolone (the pharmacologically active metabolite of prednisone) of mitogen-stimulated human lymphocyte proliferation. In this study, peripheral blood mononuclear cells (PBMC) from end stage renal disease patients were cultured with phytohemagglutinin (PHA) alone and with methylprednisolone and prednisolone individually, as well as each glucocorticoid (10(-7) mol/L) in combination with 300 ng/ml cyclosporine, 10 ng/ml tacrolimus, 25 microg/ml pentoxifylline, and 10(-7) mol/L mycophenolic acid. Under each experimental condition, the mean +/- SD % inhibition of PHA-stimulated 3H-thymidine incorporation was significantly greater with methylprednisolone than with prednisolone: methylprednisolone 55 +/- 17 versus prednisolone 28 +/- 14, p < 0.001; methylprednisolone + cyclosporine 76 +/- 18 versus prednisolone + cyclosporine 52 +/- 18, p < 0.001; methylprednisolone + tacrolimus 74 +/- 18 versus prednisolone + tacrolimus 50 +/- 20, p = 0.001; methylprednisolone + mycophenolic acid 69 +/- 14 versus prednisolone + mycophenolic acid 46 +/- 15, p < 0.001. These results confirm and extend previous observations and suggest that methylprednisolone might be more effective than prednisone in treatment protocols used to suppress allograft rejection.

Use of a simple light absorbance assay to measure lymphocyte proliferation.

The proliferative response of human lymphocytes to stimuli such as foreign histocompatibility antigens or mitogens is generally assessed by measuring the amount of tritiated thymidine which the cells incorporated in culture. In this paper, the possibility of assessing lymphocyte proliferation and viability by an empirical assay, using measurement of light absorbance on a ELISA reader in the yellow wave length (450 nm/air-550 nm/air), has been studied. The correlation of these measurements with a colormetric viability assay using MTS/PMS, with tritiated thymidine incorporation and with trypan blue exclusion viability counting, was determined. The results showed that the light absorbance assay correlated well with cell proliferation during 48-120 hours culture period and with cell viability after a 72 hour period. The MTS/PMS colormetric assay as well as trypan blue exclusion cell counting confirmed that the light absorbance assay was not merely caused by dead cells. This data confirm that the light absorbance assay is sufficiently sensitive to low levels of proliferation to allow detection of such responses at least as effectively as thymidine incorporation. The light absorbance assay procedure avoids the expense, time and hazards associated with scintillation counting, and is simple to perform without the necessity for reagents and preparative steps required by other assays.

Effects of tacrolimus on hyperlipidemia after successful renal transplantation: a Southeastern Organ Procurement Foundation multicenter clinical study.

BACKGROUND: Tacrolimus has been shown to have a less adverse effect on the lipid profiles of transplant patients when the drug is started as induction therapy. In order to determine the effect tacrolimus has on lipid profiles in stable cyclosporine-treated renal transplant patients with established hyperlipidemia, a randomized prospective study was undertaken by the Southeastern Organ Procurement Foundation. METHODS: Patients of the 13 transplant centers, with cholesterol of 240 mg/dl or greater, who were at least 1 year posttransplant with stable renal function, were randomly assigned to remain on cyclosporine (control) or converted to tacrolimus. Patients converted to tacrolimus were maintained at a level of 5-15 ng/ml, and control patients remained at their previous levels of cyclosporine. Concurrent immunosuppressants were not changed. Levels of total cholesterol, triglycerides, total high-density lipoprotein, low-density lipoprotein (LDL), very-low-density lipoprotein, and apoproteins A and B were monitored before conversion and at months 1, 3, and 6. Renal function and glucose control were evaluated at the beginning and end of the study (month 6). RESULTS: A total of 65 patients were enrolled; 12 patients failed to complete the study. None were removed as a result of acute rejection or graft failure. Fifty-three patients were available for analysis (27 in the tacrolimus group and 26 controls). Demographics were not different between groups. In patients converted to tacrolimus treatment, there was a -55 mg/dl (-16%) (P=0.0031) change in cholesterol, a -48 mg/dl (-25%) (P=0.0014) change in LDL cholesterol, and a -36 mg/dl (-23%) (P=0.034) change in apolipoprotein B. There was no change in renal function, glycemic control, or incidence of new onset diabetes mellitus in the tacrolimus group. CONCLUSION: Conversion from cyclosporine to tacrolimus can be safely done after successful transplantation. Introduction of tacrolimus to a stable renal patient does not effect renal function or glycemic control. Tacrolimus can lower cholesterol, LDL, and apolipoprotein B. Conversion to tacrolimus from cyclosporine should be considered in the treatment of posttransplant hyperlipidemia.