Biotransformation of Endocrine-Disrupting Compounds in Groundwater: Bisphenol A, Nonylphenol, Ethynylestradiol and Triclosan by a Laccase Cocktail from Pycnoporus sanguineus CS43.

The biodegradation of organic compounds present in water at trace concentration has become a critical environmental problem. In particular, enzymatic oxidation by fungal laccases offers a promising alternative for efficient and sustainable removal of organic pollutants in water. In this work, the biocatalytic ability of laccases from the Pycnoporus sanguineus CS43 fungus was evaluated. A filtered culture supernatant (laccase cocktail) evidenced an enhanced biotransformation capability to remove common endocrine-disruptor compounds (EDCs), such as bisphenol A, 4-nonylphenol, 17-α-ethynylestradiol and triclosan. A biodegradation of around 89-100 % was achieved for all EDCs using synthetic samples (10 mg L-1) and after the enzymatic treatment with 100 U L-1 (50.3 U mg -1). The biodegradation rates obtained were fitted to a first order reaction. Furthermore, enzymatic biocatalytic activity was also evaluated in groundwater samples coming from northwestern Mexico, reaching biotransformation percentages between 55 and 93 % for all tested compounds. As far as we know this is the first study on real groundwater samples in which the enzymatic degradation of target EDCs by a laccase cocktail from any strain of Pycnoporus sanguineus was evaluated. In comparison with purified laccases, the use of cocktail offers operational advantages since additional purification steps can be avoided.

Combined islet and hematopoietic stem cell allotransplantation: a clinical pilot trial to induce chimerism and graft tolerance.

To prevent graft rejection and avoid immunosuppression-related side-effects, we attempted to induce recipient chimerism and graft tolerance in islet transplantation by donor CD34+hematopoietic stem cell (HSC) infusion. Six patients with brittle type 1 Diabetes Mellitus received a single-donor allogeneic islet transplant (8611 +/- 2113 IEQ/kg) followed by high doses of donor HSC (4.3 +/- 1.9 x 10(6) HSC/kg), at days 5 and 11 posttransplant, without ablative conditioning. An 'Edmonton-like' immunosuppression was administered, with a single dose of anti-TNFalpha antibody (Infliximab) added to induction. Immunosuppression was weaned per protocol starting 12 months posttransplant. After transplantation, glucose control significantly improved, with 3 recipients achieving insulin-independence for a short time (24 +/- 23 days). No severe hypoglycemia or protocol-related adverse events occurred. Graft function was maximal at 3 months then declined. Two recipients rejected within 6 months due to low immunosuppressive trough levels, whereas 4 completed 1-year follow-up with functioning grafts. Graft failure occurred within 4 months from weaning (478 +/- 25 days posttransplant). Peripheral chimerism, as donor leukocytes, was maximal at 1-month (5.92 +/- 0.48%), highly reduced at 1-year (0.20 +/- 0.08%), and was undetectable at graft failure. CD25+T-lymphocytes significantly decreased at 3 months, but partially recovered thereafter. Combined islet and HSC allotransplantation using an 'Edmonton-like' immunosuppression, without ablative conditioning, did not lead to stable chimerism and graft tolerance.

Six-year clinical effect of donor bone marrow infusions in renal transplant patients.

BACKGROUND: To date, several single- and multicenter clinical trials have attempted to induce specific immunological unresponsiveness using donor bone marrow cell infusions to augment solid organ transplantation, but the outcomes have not been definitive. METHODS: Between September 1994 and May 1998, 63 cadaver (CAD) renal transplant recipients of either one or two postoperative donor bone marrow cell (DBMC) infusions were prospectively compared with 219 non-infused controls given equivalent immunosuppression. There was at least a 1 HLA DR antigen match present between donors and recipients. The immunosuppressive regimen included a 10-day course of OKT3 induction, and tacrolimus, mycophenolate mofetil, and methylprednisolone maintenance. A total 7.01x10(8)+/-1.9x10(8) (SD) DBMC/kg was infused into the CAD recipients on either days 4 and 11 (n=42) or one half of that dose on day 4 (n=21) postoperatively. Clinical follow-up has ranged from 2.9 to 6.3 years (mean, 4.7 years). Studies were also performed of humoral immunity and quantitative cellular chimerism. RESULTS: There is clear-cut equivalence in immunosuppressive dosaging and in the other major demographic variables in both groups. However, only 2/63 DBMC recipients had biopsy-proven chronic rejection, whereas 41/219 showed chronic rejection in the controls (P = <0.01). In both groups, mortality was not associated with rejection. The actuarial graft survival at 6.3 years in the CAD DBMC group was 84.3% compared with 72.2% in the control group (not statistically significant). However, if death with a functioning graft was excluded, graft survival was 94.1% in the DBMC group and 79.8% in the controls (P=0.039). Forty patients in the control group continue to have deteriorating renal function (increasing serum creatinine concentrations to 2 mg/dl and higher), compared with 2 patients in the DBMC group (P=0.04). In the DBMC group, chimerism in iliac crest marrow aspirates has increased 3-fold in yearly sequential measurements between 1 and 4 years postoperatively averaging 1.3+/-0.36% (SE) most recently. This has not occurred in the controls. CONCLUSIONS: There now appears to be more solid long-term evidence, in kidney transplant recipients prospectively receiving DBMC infusions, of an improvement in long-term graft survival, and of the degree of chimerism positively correlating with the absence of graft loss.

Donor bone marrow-derived chimeric cells present in renal transplant recipients infused with donor marrow. I. Potent regulators of recipient antidonor immune responses.

BACKGROUND: Even though a number of transplant centers have adopted donor-specific bone marrow cell (DBMC) infusions to enhance donor cell chimerism, to date there has been no direct evidence linking chimerism with tolerance induction in human organ transplant recipients. METHODS: Cells of donor phenotype were isolated 1 year postoperatively from the peripheral blood lymphocytes and iliac crest bone marrow of 11 living-related-donor (LRD) renal transplant recipients, who had received perioperative donor bone marrow cell infusions. These recipient-derived donor (RdD) cells were characterized phenotypically by flow cytometric analysis and functionally as modulators in mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) assays. RESULTS: The yield of RdD cells ranged from 0.1 to O.9% of the starting material with the majority being TcRalphabeta, CD3 positive T cells, a substantial percentage of which coexpressed CD28. At 1 year posttransplant almost 50% of the LRD-kidney/DBMC recipients tested so far exhibited donor-specific unresponsiveness in MLR (7/17) and CML (6/13) reactions and this trend was further enhanced at 23 years. In the recipients with residual positive antidonor immune responses, the RdD cells inhibited recipient antidonor MLR and CML responses significantly more strongly than freshly isolated and similarly treated iliac crest bone marrow cells from the donor. RdD cells also inhibited the MLR of the recipient to third party allogeneic stimulator cells; however, this nonspecific effect was significantly weaker than specific inhibition. We also established long-term bone marrow cultures stimulated every 2 weeks with irradiated alogeneic feeder cells, that had similar functional properties thus possibly providing us with an in vitro correlate the RdD cells. CONCLUSIONS: These results clearly support the notion that the infused donor cells play a positive role in the induction and/or maintenance of transplant tolerance.

Involvement of multiple subpopulations of human bone marrow cells in the regulation of allogeneic cellular immune responses.

BACKGROUND: The identity of the cells in the human bone marrow that function as effective regulators of in vitro and possibly in vivo cellular immune responses is not well established. METHODS: Cell subpopulations were isolated from cadaver donor vertebral-body bone marrow cells (DBMC) by using immuno-magnetic microbeads and were tested as inhibitors (modulators) in cell-mediated lympholysis (CML) and mixed lymphocyte reaction (MLR) responses of normal peripheral blood lymphocytes stimulated with irradiated cadaver donor spleen cells. RESULTS: Compared with spleen cells as controls, un-irradiated T-cell depleted DBMC inhibited both the MLR and CML responses of allogeneic responder cells in a dose dependent manner (as in our previous reports). The inhibition was also mediated by a number of purified subpopulations including pluripotent CD34+ stem cells, and their CD34 negative early progeny of both lymphoid and myeloid lineages. These included DBMC enriched for non-T-cell lymphoid precursors (NT-LP/DBMC; i.e., DBMC depleted of CD3, CD15, and glycophorin-A positive cells) and DBMC positively selected for CD38+, CD2+, CD5+, and CD1+ lymphoid cells (all were depleted of CD3+ cells) as well as CD33+ (but CD15 negative) myeloid precursors. However, positively selected CD19+ B-cells and CD15+ myeloid cells did not inhibit the MLR and CML responses. The NT-LP/DBMC that had been repeatedly stimulated with irradiated allogeneic peripheral blood lymphocytes caused the strongest inhibition of the MLR and CML responses of the same allogeneic cells with 200 times fewer modulator cells needed than uncultured DBMC (P<0.001). Flow cytometric analysis revealed that majority of cells in these cell lines had become CD3+ TcR-alphabeta+ CD4+ and CD28+ cells. CONCLUSION: A variety of less differentiated cells of various lineages residing in the human bone marrow are immunoregulatory in vitro. Among them, there is at least one subset that can undergo differentiation in vitro into regulatory T cells that can be maintained in long-term cultures.

Kidney transplantation at the University of Miami.

Of the 1,679 renal allografts performed at the University of Miami between January 1, 1979 and October 31, 1999, 1,154 were from cadaver donors (CAD), 515 were from living-related donors (LRD), and 10 were from living-unrelated donors. The 3 ethnic groups: Black Caribbean-African-American, Hispanic, and others were almost equally represented among recipients. Recipient ages ranged between 1-83 years. In the CAD group, HLA matching was emphasized so that no patient received a kidney with less than one DR match, and for the entire series a mean of 2.59 of 6 HLA antigens were matched between donors and recipients. Overall actuarial 20-year patient and graft survival rates were 65.3% and 30.7%, respectively, with 69.2% patient and 38.5% graft survival rates for LRD, and 65.6% patient and 29.0% graft survival rates for CAD recipients. Several factors adversely affected long-term graft outcome. African-Americans had an overall 20-year graft survival rate of 13.6% compared with 34% for non African-Americans (p < 0.001) (not dependent on patient survival). Diabetic patients had an overall 20-year graft survival rate of 13.5% versus 34.2% for non-diabetics (primarily dependent on patient survival). In the category of non African-American, non-diabetic patients under age 36 (n = 412), the 20-year patient survival rates in the LRD and CAD groups were 85.0% and 79.3%, respectively, and the graft survival rates were 55.7% and 46.5%, respectively. This differed markedly from the results for the entire series.

Continuing observations on the regulatory effects of donor-specific bone marrow cell infusions and chimerism in kidney transplant recipients.

BACKGROUND: Continued follow-up of a series of donor bone marrow cell (DBMC)-infused first cadaver renal transplant recipients is described (n=58), now at a 36-month actuarial time point postoperatively. Serial polymerase chain reaction-flow cytometry (PCR-Flow) and cellular immune assays of iliac crest bone marrow aspirates and peripheral blood have begun to be compared with concomitantly transplanted recipients of living-related donor (LRD) kidneys and donor marrow infusions given the same immunosuppressive regimen (n=16). There have also been comparisons (36 months) with 188 controls transplanted concomitantly, i.e., recipients of first cadaver kidney transplants, who did not receive bone marrow. METHODS: Each group was given equivalent immunosuppressive regimens of OKT3 anti-T cell induction and maintenance tacrolimus, mycophenolate mofetil, and methylprednisolone. Actuarial patient and graft survival have been 96% and 93%, respectively, in the controls and 91% and 91%, respectively, in the DBMC-infused recipients. Trough levels of tacrolimus were significantly lower in the DBMC-infused group. RESULTS: In PCR-Flow measurements, in peripheral blood up to 6 months postoperatively, there were higher levels of chimerism, i.e., in the total number of donor cells, as well as the donor CD3+ and CD34+ subsets in the LRD recipients administered DBMC infusions, compared with cadaver DBMC recipients, supporting the notion of a positive effect of histocompatibility on chimerism levels. In PCR-Flow measurements of recipient iliac crest bone marrow aspirates as in previous studies on peripheral blood, early acute rejection episodes (<1 month) were found to be associated with a later (6-14 months) decrease in donor cell lineage chimerism. However, a trend toward recovery of chimeric levels occurred by 21-28 months in a second iliac crest marrow aspirate 1 year after the first aspirate in the DBMC-infused recipients who experienced such early rejection episodes. This was in contrast to the controls in whom there were sustained low levels of iliac crest bone marrow chimerism at both the earlier and later intervals (i.e., no chimeric recovery), with 17/183 surviving controls progressing into chronic rejection. This has not yet been seen in the DBMC-infused group (0/54). In in vitro observations on cellular immune reactivity at 1 year postoperatively, decreased peripheral blood lymphocyte proliferative reactions were seen in response to phytohemagglutinin and Staph-A mitogens, as well as to cytomegalovirus and Epstein-Barr viral protein antigens in the DBMC-infused group versus the controls. Chronic immunosuppression did not seem to effect a vigorous in vitro inhibitory (regulatory) activity of bone marrow taken from these transplant recipients 2 years postoperatively in mixed lymphocyte culture and cell-mediated lympholysis reactions, using allogeneic responding cells from "normal" laboratory volunteers. Autologous peripheral blood lymphoproliferative responses to phytohemagglutinin and Staph-A mitogens, as well as to cytomegalovirus and Epstein-Barr virus protein antigens, were also regulated by either organ donor (non-immunosuppressed) bone marrow cells or by transplant recipient (immunosuppressed) bone marrow cells. What appeared to be disparate between the DBMC-infused and control groups (both immunosuppressed) was the trend for the (autologous) bone marrow suppressive effect on antiviral lymphoproliferative responses, to be stronger in the DBMC-infused group, who also had significantly (>one order of magnitude) higher levels of chimerism (P=0.01). CONCLUSIONS: It is concluded that the establishment of a chimeric state in DBMC-infused recipients, albeit of relatively low magnitude (approximately 1% at 2 years in recipient iliac crest bone marrow), has had a definite regulatory effect on immune responses. These results, therefore, add weight to the "causal" horn of the dilemma as to whether donor cell chimerism is a cause or an effect of

Donor peripheral blood stem cell infusions in recipients of living-related liver allografts.

BACKGROUND: In this pilot study, donor peripheral blood stem cell (DPBSC) infusions were performed in three recipients of living-related liver transplants (LRLT). METHODS: DPBSCs were obtained by leukapheresis after mobilization with granulocyte-colony-stimulating factor (Filgrastim). Donor leukapheresis was performed on the 5th postoperative day, and half of the DPBSCs were infused into the recipient on the day of collection. The second half of the pheresed product was cryopreserved for delayed administration. RESULTS: Results from preliminary studies of chimerism in LRLT recipients, at 20 weeks posttransplant, suggested that the levels of donor cells detected in LRLT recipients treated with DPBSC infusions may be higher than those observed for recipients of cadaver donor liver allografts and vertebral body marrow infusions. CONCLUSIONS: The results of this pilot study indicate that administration of mobilized DPBSC to recipients of LRLT is a feasible procedure for both donor and recipient.

The effects of chimeric cells following donor bone marrow infusions as detected by PCR-flow assays in kidney transplant recipients.

40 recipients of first cadaver kidney transplants were given perioperative donor vertebral bone marrow infusions (DBMC), compared with 100 controls who did not receive donor bone marrow. The immunosuppressive regimen included OKT3, Tacrolimus, and steroid maintenance therapy, and, in some patients, newly introduced mycophenolate mofetil. This report describes the 24-mo actuarial follow-up and several immunological monitoring studies including sequential measurements of donor bone marrow lineage subset chimerism by the recently reported PCR-flow assay. This is a sensitive in situ PCR detection system for donor versus recipient histocompatibility genes as well as cell surface CD epitope markers using flow cytometry. The results indicate (a) the stabilization of the donor CD3+ and CD34+ cells in recipient peripheral blood at levels below 1% between 6 mo and 1 yr postoperatively, with a 10-fold higher level of donor cell chimerism of these lineages in recipient iliac crest marrow; (b) significantly lower levels of chimerism in peripheral blood up to 6 mo postoperatively in patients who had early acute (reversible) rejection episodes compared with those who did not; (c) a higher degree of chimerism seen in patients who were class II MHC HLA DR identical with their donors; (d) the identification of a high proportion of the donor bone marrow derived CD3 dimly staining subset of T cells (to which regulatory functions have been ascribed) in recipient peripheral blood and especially in recipient bone marrow; and (e) an unexpectedly increased susceptibility to clinically significant infections (primarily viral), and even death in the DBMC-infused group, compared with controls, but no graft losses because of rejection in the DBMC-infused group. Mixed lymphocyte culture assays showed a trend toward a greater number of nonspecifically low reactors in the DBMC group, as well as a greater number of nonspecifically high reactors in the controls (P = 0.058). The autologous mixed lymphocyte reaction also indicated a trend towards nonspecific immune activation in the DBMC group. Finally, anti-cytomegaloviral IgG antibody reactivity was significantly inhibited in the DBMC group 4-6 mo postoperatively (P = < 0.05). In the controls, there were no donor cell lineages detected by PCR-flow in the peripheral blood. These rather unexpected findings, indicating a more depressed cellular and humoral immune capacity in the DBMC cadaver kidney transplant recipients in this relatively early follow-up period, are discussed relevant to chimerism, MHC restriction, and suppressor activity brought about by specialized DBMC subsets, which still need to be defined.