Biomedical subjects

C Ricordi

Publications and source records attributed to C Ricordi.

At least 91 records · Page 5Linked to original sources

PubMed1998

Efficient ex vivo inhibition of perforin and Fas ligand expression by chimeric tRNA-hammerhead ribozymes.

Graft-versus-host disease (GVHD) is a feared complication of allogeneic bone marrow transplantation. Research in rodent models has linked perforin and Fas ligand (FasL), two components of independent lytic pathways, with the induction of GVHD. In this study we characterized two hammerhead ribozymes that cleave their target perforin and Fas ligand RNAs with high efficiency in CTLL-2 cells. The perforin and Fas ligand ribozymes were expressed from a tRNA-directed RNA polymerase III promoter that was inserted in an episomal multicopy plasmid derived from papilloma virus. Chimeric anti-perforin and anti-FasL tRNA-ribozymes had sequences engineered in order to have specific secondary structure effects. These sequence modifications allow the formation of a 5' --> 3' stem structure and also place the ribozyme in a flexible bulge region that keeps the ribozyme separated from the tRNA domain. Northern and RT in situ PCR analyses showed high levels of transcription and efficient transportation to the cytoplasm. The expression of perforin and FasL in CTLL-2 cells was significantly reduced as assessed by RNA and protein analyses.

Animals↗

PubMed1998

Cellular immune responses of human cadaver donor bone marrow cells and their susceptibility to commonly used immunosuppressive drugs in transplantation.

BACKGROUND: The cascade of immunological effects brought about by donor bone marrow cell (DBMC) infusions in human organ transplantation, especially in the context of continuous pharmacologic immunosuppression, is not fully understood. Yet, in inbred rodents and even primates, administration of specific bone marrow cells has caused a state of acquired immunologic tolerance. METHODS: In vitro mixed lymphocyte culture (MLC) and cell-mediated lympholysis (CML) culture systems were used to compare the responding and regulatory properties of DBMC and individual bone marrow cell subsets versus spleen cells in the presence or absence of pharmacologic immunosuppression. RESULTS: In the absence of immunosuppressive drugs, the DBMC proliferated in MLC and in response to phytohemagglutinin, but to a lower magnitude than donor spleen cells. In CML assays, DBMC failed to function as cytotoxic cells. Removal of both CD3+ and CD34+ cells together (not just singly) had to occur for complete abrogation of the proliferative response of DBMC evoked in the presence of allogeneic stimulating cells. Testing several experimental variables using flow cytometric analysis led to the conclusion that when purified DBMC CD34+ cells were placed in coculture with irradiated allogeneic peripheral blood mononuclear cells, such CD34+ cells give rise both to CD3- TCRalphabeta+ as well as to dimly staining CD3+ TCRalphabeta+ cells. Low pharmacologic concentrations of tacrolimus/cyclosporine (CsA) and mycophenolic acid (MPA) singly or in combination had no effect on the spontaneous proliferation of DBMC and had significantly less inhibitory activity on MLC responses of DBMC and its purified CD3+ or CD34+ subpopulations, compared with the responses of spleen cells. Moreover, the previously described regulatory effects of DBMC on the MLC responses of peripheral blood or splenic responding cells were not inhibited by these immunosuppressive drugs. CONCLUSIONS: Taken together, these results support the notion that in vitro DBMC subpopulations, which proliferate as responding cells in co-culture with x-irradiated allogeneic cells and which cause regulatory effects when added as a third component to MLC reactions, seem to be culture-generated lymphoid cell lineage(s) progeny of CD34+ cells. This possibly includes unique CD3+ "primitive" (dimly staining) T cells, which are not as inhibited in their function by tacrolimus/CsA and MPA, as are postthymic (splenic) T cells.

Antigens, CD34↗

PubMed1998

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

Bone Marrow Cells↗

PubMed1998

KSA antigen Ep-CAM mediates cell-cell adhesion of pancreatic epithelial cells: morphoregulatory roles in pancreatic islet development.

Cell adhesion molecules (CAMs) are important mediators of cell-cell interactions and regulate cell fate determination by influencing growth, differentiation, and organization within tissues. The human pancarcinoma antigen KSA is a glycoprotein of 40 kD originally identified as a marker of rapidly proliferating tumors of epithelial origin. Interestingly, most normal epithelia also express this antigen, although at lower levels, suggesting that a dynamic regulation of KSA may occur during cell growth and differentiation. Recently, evidence has been provided that this glycoprotein may function as an epithelial cell adhesion molecule (Ep-CAM). Here, we report that Ep-CAM exhibits the features of a morphoregulatory molecule involved in the development of human pancreatic islets. We demonstrate that Ep-CAM expression is targeted to the lateral domain of epithelial cells of the human fetal pancreas, and that it mediates calcium-independent cell-cell adhesion. Quantitative confocal immunofluorescence in fetal pancreata identified the highest levels of Ep-CAM expression in developing islet-like cell clusters budding from the ductal epithelium, a cell compartment thought to comprise endocrine progenitors. A surprisingly reversed pattern was observed in the human adult pancreas, displaying low levels of Ep-CAM in islet cells and high levels in ducts. We further demonstrate that culture conditions promoting epithelial cell growth induce upregulation of Ep-CAM, whereas endocrine differentiation of fetal pancreatic epithelial cells, transplanted in nude mice, is associated with a downregulation of Ep-CAM expression. In addition, a blockade of Ep-CAM function by KS1/4 mAb induced insulin and glucagon gene transcription and translation in fetal pancreatic cell clusters. These results indicate that developmentally regulated expression and function of Ep-CAM play a morphoregulatory role in pancreatic islet ontogeny.

Adult↗

PubMed1998

Islet transplantation: present and future perspectives.

Islet cell transplantation can potentially normalize blood glucose levels and stop the progression of clinical complications, and if the transplant is done early in the course of the disease complications may be prevented. Remarkable progress has been made in recent years and islet cell transplantation has resulted in normalization of metabolic control in several patients with Type 1 diabetes in the absence of hypoglycemia. Only a few patients, however, have achieved insulin independence. Issues relating to islet cell engraftment within the liver, prevention of rejection and recurrent autoimmunity, and identification of alternative immunosuppressive drugs that do not adversely affect islet cell function remain to be solved. Thus far, the need for chronic, generalized immunosuppression to prevent rejection of the islets has limited the indication to those patients who have already received another transplant or to those who simultaneously receive islets and another organ (generally a kidney). Identification of immunointervention protocols that allow for engraftment in the absence of deleterious effects on the islets and prevent rejection and recurrent autoimmunity would make this procedure suitable for all patients, including children who have not yet developed long-term complications of the disease.

Clinical Trials as Topic↗

PubMed1998