Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation

BACKGROUND: Marrow stromal cells (MSC) can differentiate into multiple mesenchymal tissues. To assess the feasibility of human MSC transplantation, we evaluated the in vitro immunogenicity of MSC and their ability to function as alloantigen presenting cells (APC). METHODS: Human MSC were derived and used in mixed cell cultures with allogeneic peripheral blood mononuclear cells (PBMC). Expression of immunoregulatory molecules on MSC was analyzed by flow cytometry. An MSC-associated suppressive activity was analyzed using cell-proliferation assays and enzyme-linked immunoassays. RESULTS: MSC failed to elicit a proliferative response when cocultured with allogeneic PBMC, despite provision of a costimulatory signal delivered by an anti-CD28 antibody and pretreatment of MSC with gamma-interferon. MSC express major histocompatibility complex (MHC) class I and lymphocyte function-associated antigen (LFA)-3 antigens constitutively and MHC class II and intercellular adhesion molecule (ICAM)-1 antigens upon gamma-interferon treatment but do not express CD80, CD86, or CD40 costimulatory molecules. MSC actively suppressed proliferation of responder PBMC stimulated by third-party allogeneic PBMC as well as T cells stimulated by anti-CD3 and anti-CD28 antibodies. Separation of MSC and PBMC by a semipermeable membrane did not abrogate the suppression. The suppressive activity could not be accounted for by MSC production of interleukin-10, transforming growth factor-beta1, or prostaglandin E2, nor by tryptophan depletion of the culture medium. CONCLUSIONS: Human MSC fail to stimulate allogeneic PBMC or T-cell proliferation in mixed cell cultures. Unlike other nonprofessional APC, this failure of function is not reversed by provision of CD28-mediated costimulation nor gamma-interferon pretreatment. Rather, MSC actively inhibit T-cell proliferation, suggesting that allogeneic MSC transplantation might be accomplished without the need for significant host immunosuppression.     

Regulation of osteoblast proliferation by leukemia inhibitory factor.

We recently showed that leukemia inhibitory factor (LIF) stimulates 45Ca release from neonatal mouse calvariae in vitro and that it increases DNA and protein synthesis in this model. To elucidate further the actions of LIF on bone we now report the effects of this cytokine on DNA synthesis and cell proliferation in isolated fetal rat osteoblasts and in the osteogenic sarcoma cell line, UMR-106. In both actively growing and growth-arrested rat osteoblasts, LIF stimulated [3H]thymidine incorporation in a dose-dependent manner. The increase in DNA synthesis was time dependent, was associated with an increase in the number of osteoblasts, and was not blocked by indomethacin. LIF-treated cells showed reduced [3H]thymidine incorporation in comparison with control, as they approached confluence, possibly because of the increased cell density in the LIF-treated cultures. In UMR-106 cells, treatment with LIF inhibited [3H]thymidine incorporation in both actively growing and growth-arrested cultures. The effect was dose dependent and sustained with time. There was a corresponding decrease in cell numbers. It is concluded that although LIF causes an early stimulation of proliferation in isolated osteoblasts, it has opposing effects on UMR-106 cells. It is not possible to determine which of these effects is more relevant to the actions of LIF in vivo. The demonstration of a LIF effect on both these cell types, however, provides further evidence that this cytokine acts directly on osteoblasts.     

Parathyroid transplantation: fate of a long-term allograft in man.

Previously our group reported clinical, histological, and biochemical evidence of parathyroid allograft survival in a patient transplanted sequentially with renal and parathyroid tissues from the same donor. After 30 months of function, both grafts were concomitantly rrjected. The prolonged parathyroid allograft survival described in certain experimental animals has been attributed to the tissue's being "immunologically privileged." Our current clinical observations do not support this hypothesis.     

Sarpogrelate inhibits serotonin-induced proliferation of porcine coronary artery smooth muscle cells: implications for long-term graft patency

Background. Serotonin can induce proliferation of vascular smooth muscle cells. We assessed the ability of a specific serotonin receptor antagonist, sarpogrelate, to inhibit proliferation of cultured porcine coronary artery smooth muscle cells.

Methods. Cell proliferation and mitotic activity were measured using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide. To determine the effect of sarpogrelate on DNA (deoxyribonucleic acid), RNA (ribonucleic acid), and protein synthesis, radioactive incorporation of ³H-thymidine, ³H-uridine, and ³H-phenylalanine, respectively, was used. Synthesis of DNA was also assessed by flow cytometry with propidium iodide as a fluorochrome.

Results. Serotonin, platelet-derived growth factor, endothelin, and angiotensin II all induced proliferation of porcine coronary artery smooth muscle cells. Sarpogrelate specifically inhibited the serotonin-induced cytokine trigger but did not influence platelet-derived growth factor–, endothelin–, or angiotensin II–induced cell proliferation. Sarpogrelate inhibited the serotonin-induced increase in intracellular free ionized calcium concentration, prevented mitogen-activated protein kinase activation, and down-regulated expression of the protooncogenes c-fos and c-jun. Sarpogrelate acted at the G₁ phase of the cell cycle.

Conclusions. These data suggest that sarpogrelate could be used as a therapeutic agent to inhibit serotonin-induced neointimal hyperplasia and improve patency of coronary artery bypass grafts.     

A 12-day course of FK506 allows long-term acceptance of semi-identical liver allograft in inbred miniature swine

BACKGROUND: Spontaneous tolerance to liver allograft has been reported previously in outbred pig models, but the lack of genetic background did not allow to analyze the impact of the major histocompatibility complex (MHC) on tolerance induction. A model of semi-identical liver allograft was therefore developed in inbred miniature swine in order to mimic the clinical situation of living related liver transplant (parent into infant) and to study a protocol for inducing tolerance to liver allograft. METHODS: SLAdd (class Id/d, class IId/d) pigs received orthotopic liver allograft from heterozygous SLAcd (class Ic/d, class IIc/d) miniature swine. Eight animals did not receive immunosuppression. Fourteen SLAdd animals had a 12-day course of FK506 and were divided in two subgroups. In subgroup FK-1, six pigs received a daily intramuscular injection of FK506 at 0.1-0.4 mg/kg, in order to reach daily trough levels between 7 and 20 ng/ml; in subgroup FK-2, eight additional animals received two daily injections of FK506 at 0.05 mg/kg regardless of the daily trough levels. Graft survival, liver biological tests, histology, cellular and humoral immune responses, as well as detection of microchimerism were assessed in all groups. RESULTS: All untreated animals rejected their allograft and died within 28.1 +/- 9.5 days. These rejector animals developed a significant anti-donor cellular and humoral immune response. No peripheral or lymphoid tissue microchimerism was detected in this group. In contrast, long-term survival was obtained in five FK-treated animals (112, 154, 406, 413, and 440 days), whereas several pigs died with a normal allograft function from either overimmunosuppression or intercurrent causes. All FK-treated pigs developed a specific anti-donor unresponsiveness in both cell mediated lymphocytotoxicity and mixed lymphocyte reaction and did not develop anti-donor alloantibodies. The study of the anti-donor immune response by mixed lymphocyte reaction, during the first postoperative week, demonstrated a specific anti-donor unresponsiveness in the peripheral blood from the first posttransplant day. Although microchimerism was detectable in the peripheral blood for several postoperative weeks (maximum 10 weeks) in FK-treated animals, donor cells or DNA were not detected during the long-term follow-up in peripheral blood or lymphoid tissues. CONCLUSIONS: Spontaneous tolerance to semi-identical orthotopic liver allograft did not occur, whereas a 12-day course of FK506 allowed long-term graft acceptance. All FK-treated animals developed in vitro signs of specific immune unresponsiveness and transient peripheral microchimerism. The specific anti-donor cellular unresponsiveness occurred on the first postoperative day after surgery and was of long-term duration. The study of the early immunological events in this model could be of major importance regarding clinical living related liver transplantation.     

Kidney allograft offers: Predictors of turndown and the impact of late organ acceptance on allograft survival

There is growing interest in understanding patterns of organ acceptance and reducing discard. Little is known about how donor factors, timing of procurement, and geographic location affect organ offer decisions. We performed a retrospective cohort study of 47 563 deceased donor kidney match‐runs from 2007 to 2013. Several characteristics unrelated to allograft quality were independently associated with later acceptance in the match‐run: Public Health Service increased‐risk donor status (adjusted odds ratio [aOR] 2.49, 95% confidence interval [CI] 2.29‐2.69), holiday or weekend procurement (aOR 1.11, 95% CI 1.07‐1.16), shorter donor stature (aOR 1.53 for <150 cm vs reference >180 cm, 95% CI 1.28‐1.94), and procurement in an area with higher intensity of market competition (aOR 1.71, 95% CI 1.62‐1.78) and with the longest waiting times (aOR 1.41, 95% CI 1.34‐1.49). Later acceptance in the match‐run was associated with delayed graft function but not all‐cause allograft failure (adjusted hazard ratio 1.01, 95% CI 0.96‐1.07). Study limitations include a lack of match‐run data for discarded organs and the possibility of sequence inaccuracies for some nonlocal matches. Interventions are needed to reduce turndowns of viable organs, especially when decisions are driven by infectious risk, weekend or holiday procurement, geography, or other donor characteristics unrelated to allograft quality.     

Preoperative low-dose irradiation promotes long-term allograft acceptance and induces regulatory T cells in a porcine model of pulmonary transplantation

BACKGROUND: A simplified conditioning protocol including single-dose preoperative thymic and low-dose whole body irradiation with or without subsequent donor bone marrow transplantation (BMTx) can be applied in porcine lung transplantation. We hypothesized that this protocol would prolong allograft survival. METHODS: Left-sided single lung transplantation from major histocompatibility complex (MHC)-mismatched donors was performed in 27 minipigs. Recipients received whole body (1.5 Gy) and thymic irradiation (7 Gy) before transplantation (IRR group, n=6), intravenous immunosuppression with methylprednisolone, cyclosporine, and azathioprine for 27 postoperative days (IS group, n=5) or both (IRR+IS group, n=10). BMTx group recipients were treated with irradiation, immunosuppression and a donor bone marrow infusion on postoperative day 1. Peripheral blood leukocyte phenotype and donor cell chimerism were monitored by flow cytometry. Purified CD25+ T cells from long-term survivors or rejecting animals were used for in vitro MLR suppression assays. RESULTS: Median graft survival was: IRR 12 days, IS 55 days, IRR+IS 239 days, and BMTx 80 days (P<0.0001). Early peripheral blood macrochimerism was substantial in both the IRR+IS and the BMTX group but was lost in all groups after day 80. The frequency and suppressive function of CD4+CD25+ T cells were enhanced in IRR+IS group long-term survivors. CONCLUSION: Although donor bone marrow infusion was not beneficial in our model, a substantial proportion of the animals treated with irradiation and a 28-day course of immunosuppression accepted their lung allografts long term. The mechanism involved in maintaining allograft tolerance may be based on peripheral T-cell regulation.     

Conversion to sirolimus for chronic allograft dysfunction: long-term results confirm predictive value of proteinuria.

The aim was to evaluate long-term graft survival and function after conversion to sirolimus (SRL) for chronic calcineurin inhibitor (CNI) toxicity and the predictive value of baseline proteinuria. This is a follow-up conversion study of 59 renal transplant patients with deteriorating graft function and histologic signs of CNI toxicity. Previously, baseline proteinuria <800 mg/day was identified as a short-term predictor for successful conversion. Follow-up was 5.3 +/- 0.8 (3.7-6.8) years. Patient survival was 88%, graft survival 38%. Creatinine clearance at the last follow-up was 33.7 +/- 14 ml/min, proteinuria 826 +/- 860mg/day. Baseline proteinuria <800 mg/day was associated with better graft survival. In a cox analysis including proteinuria >800 mg, glomerular filtration rate, age at conversion, chronic Banff score at conversion and time after transplantation at conversion, higher proteinuria was associated with a relative risk of graft loss of 3.98. Prognosis of chronic allograft dysfunction is poor. However, conversion to SRL remains an option for patients with low baseline proteinuria, which can slow down deterioration of graft function during a follow-up period of up to 5 years.     

Regulation of urinary bladder capacity by endogenous opioid peptides

Naloxone administered to chloralose or ketamine anesthetized cats reduced urinary bladder capacity. Successive cystometrograms revealed that naloxone in doses of 0.5 microgram./kg. to 15 micrograms./kg. i.v. reduced the volume necessary to evoke micturition by 10 to 50 per cent, respectively. The effect was maximal within a few minutes, remained constant for about 1/2 hour and returned to control values over the next 2 to 3 hours. Following return to control, subsequent doses of naloxone produced no further effect on capacity. In chloralose anesthetized animals naloxone also increased the frequency and amplitude of low amplitude pressure waves on the tonus limb of the cystometrogram. Intrathecal administration of naloxone to the sacral spinal cord did not significantly reduce the volume necessary to evoke micturition even at large doses, but did increase the amplitude of micturition contractions. These data, along with previous reports, suggest that mu receptors in the brainstem alter urinary bladder capacity, while delta receptors in the spinal cord modulate the magnitude of bladder contractions. Pharmacological manipulation of these receptor systems could provide a tool for the management of neurogenic bladder dysfunction.     

Kidney transplantation in Alport's syndrome: long-term outcome and allograft anti-GBM nephritis.

Anti-glomerular basement membrane (anti-GBM) nephritis occurring in kidneys transplanted in patients with Alport's syndrome (AS) has been reported repeatedly. Therefore, we studied graft survival and course of renal function in all 30 AS patients grafted at Hannover Medical School and compared them with non-diabetic, age and sex matched patients, transplanted on the date closest to the transplantation of the AS patient. Serum creatinine, proteinuria, urinary sediment and anti-GBM antibodies were examined in all AS patients with functioning grafts. Cases of patient or graft loss in the AS group were analyzed retrospectively. One- and five-year patient survival was 100 and 91% in AS and 89 and 78% in controls (p > 0.05, respectively). One- and five-year first graft survival was 79 and 66% in both groups. Graft histology was available in 34 biopsies obtained from 21 kidneys in 15 AS patients. Anti-GBM nephritis was not detected in any of the biopsies. No graft was lost due to anti-GBM nephritis. Anti-GBM antibodies were detectable temporarily only in one AS patient. He also had linear IgG staining in his graft GBM, but no other signs of anti-GBM nephritis. We conclude that patient survival and graft survival in AS patients following kidney transplantation is not different from non-AS patients. Allograft anti-GBM nephritis is a rare complication in patients with Alport's syndrome.     

Immunologic factors: the major risk for decreased long-term renal allograft survival

BACKGROUND: Both antigen-dependent (immunologic) and non-antigen-dependent (nonimmunologic) factors have been implicated in long-term renal allograft loss. Differentiating between these two factors is important because prevention strategies differ. METHODS: To isolate the importance of these 2 factors, we studied long-term actuarial graft survival in a cohort of adult kidney recipients who underwent transplants at a single institution between January 1, 1984 and October 31, 1998. Excluded were recipients with graft loss as a result of death with function, technical failure, primary nonfunction, and recurrent disease, leaving 1587 recipients (757 cadaver [CAD], 830 living donor [LD]) who would be at risk for graft loss secondary to both immunologic and nonimmunologic factors. These recipients were analyzed in the following 2 groups: those treated for a previous episode of acute rejection (AR) (Group1; n = 588; 328 CAD, 260 LD) and those with no AR (Group 2: n = 999; 429 CAD, 570 LD). Actuarial graft survival and causes of graft loss were determined for each group. Presumably, graft loss in Group 1 would be caused by immunologic and nonimmunologic factors; graft loss in Group 2 would be caused primarily by nonimmunologic factors. RESULTS: The 10-year graft survival rate (censored for death with function, technical failure, primary nonfunction, and recurrent disease) in Group 2 was 91%. In contrast, the 10-year graft survival rate in Group 1 was 45% (P<0.001 vs. Group 2). Causes of graft loss in Group 2 were chronic rejection in 1.8% (3.0% CAD, 0.9% LD), de novo disease, 0.4%; sepsis, 0.2%; discontinuation of immunosuppressive therapy, 0.3%; and unknown, 0.6%. In contrast, 23.8% (29.9% CAD, 16.2% LD) of recipients in Group 1 had graft loss caused by chronic rejection (P = 0.001 vs. Group 2). CONCLUSIONS: This very low incidence of chronic rejection in recipients without previous AR suggests that immunologic factors are the main determinants of long-term kidney transplant outcome; nonimmunologic factors in isolation may have only a minimal impact on long-term graft survival.     

Postoperative administration of donor B cells induces rat kidney allograft acceptance: lack of association with Th2 cytokine expression in long-term accepted grafts

BACKGROUND: Although donor leukocytes are only thought to prolong survival when administered before transplantation, recent evidence shows that they are effective at transplantation. This study aims to identify the leukocyte subset that is most active in prolonging kidney allograft survival and examine the cytokine expression in long-term acceptance. METHODS: PVG rat kidneys were transplanted to completely MHC class I and class II-mismatched DA recipients. Donor B cells or T cells, purified by negative selection, were injected i.v. at the time of transplantation. Expression of interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-gamma, and transforming growth factor-beta mRNA was measured by quantitative real-time polymerase chain reaction (PCR). Immunohistochemical analysis and terminal deoxynucleotide transferase-mediated dUTP nick-end labelling (TUNEL) staining was used to identify infiltrating cells and apoptotic cells, respectively, in sections of kidney allografts. RESULTS: Median kidney graft survival time (MST) of B cell-treated animals (n=5) was >300 days, compared with 7 days in untreated animals (n=7) (P=0.003), whereas animals treated with the same number of T cells (n=6) had a MST of 17 days (P=0.1 vs. untreated, P=0.03 vs. B cell-treated). Examination of the long-term (>300 days) accepted grafts from B cell-treated recipients showed little evidence of kidney damage but a moderate perivascular infiltrate consisting of T and B cells. This infiltrate seemed to be quiescent because there was no detectable expression of IL-2 receptors or of apoptotic cells. It produced little or no cytokine mRNA, because expression in the long-term accepted grafts was similar to levels in normal kidneys or syngeneic transplants. There was a marked increase of cytokine mRNA early after transplantation in both leukocyte-treated and untreated grafts, with more rapid appearance of IFN-gamma and IL-10 in leukocyte-treated grafts. CONCLUSIONS: Donor B cells efficiently induce long-term acceptance of transplanted kidneys in a fully MHC-mismatched rat model when administered at transplantation, by a mechanism that seems to be independent of Th2 cytokine expression within the long-term accepted graft.     

FK506 suppression of heart and liver allograft rejection. II: The induction of graft acceptance in rats.

Lewis recipients of orthotopic ACI livers had permanent graft acceptance induced with 3 doses of i.m. FK506 in the early postoperative period. They were studied 100 and 300 days posttransplantation. The recipients rejected ACI as well as Brown Norway (BN) (third-party) skin grafts, and had lymphocytes with substantial reactivity by mixed lymphocyte culture testing against ACI and third-party (BN) alloantigens. Lymphocyte subset redistribution had not occurred in the peripheral blood or spleens of these animals, and there was no evidence of suppressor cell activation by in vitro and in vivo tests. Graft-versus-host reactivity in splenic lymphoid tissues of these recipients was demonstrated with the popliteal lymph node assay. Attempts at adaptive transfer with recipient lymphocytes were unsuccessful. Heart graft acceptance was far more difficult to accomplish than liver graft acceptance, and probably was never permanent. ACI heart graft prolongation in LEW recipients after a brief induction with FK506 lasted for no more than 3 months in most animals. The temporary heart graft acceptance was specific for hearts of the original ACI donor strain but not for ACI skin. Results of studies of lymphocyte subsets and suppressor cell activity were similar to those in the liver recipients. These studies illustrate how poorly graft acceptance is understood and how badly further work is needed to clarify its mechanism.