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R Llull

Publications and source records attributed to R Llull.

30 records · Page 2Linked to original sources
PubMed1994

Cellular mechanisms of alloimmune non-responsiveness in tolerant mixed lymphocyte chimeras induced by vascularized bone marrow transplants.

It has been demonstrated that the development of stable mixed lymphocyte chimerism is associated with alloimmune tolerance induction in vascularized bone marrow transplant (VBMT) recipients. The underlying mechanisms of immune non-responsiveness in tolerant VBMT chimeras remains unclear. Our VBMT model involves the transplantation of a parental donor limb (Lewis rats) onto a hybrid (Lewis x Brown Norway) F1 recipient. Tolerogenic mechanisms and cellular immune regulation to self and host allodeterminants were investigated during the early post-transplant phase of tolerance induction. Flow cytometric analysis of sIg+-depleted experimental peripheral blood lymphocytes from tolerant VBMT recipients demonstrated low level stable mixed immune chimerism. Chimeric cells tested for responsiveness against self-LEW determinants showed activated proliferation and immune dysregulation 30 days post-transplantation. However, direct immunocytolytic activity against LEW determinants was not found. Tolerant chimeras also demonstrated elevated cellular proliferation and cytolytic responses against host-specific BN allodeterminants at 30 days. Consistent with these in vitro findings, limited clinical signs compatible with GVH reactivity were evident in vivo at this time. Following this initial period, the tolerant VBMT animals returned to normal clinical condition and remained otherwise healthy throughout the study. Consistent with these results, VBMT chimeras then showed declining proliferative responses from the elevated values seen at 30 days against self-LEW determinants. Proliferative and immunocytolytic responses also decreased against host-specific BN allodeterminants from peak levels at 30 days. In conclusion, these results provide evidence that the initial phases of tolerance induction in VBMT chimeras consist of self- and alloimmune regulation that follow an early period of immune dysregulation. Sequential phases of immune dysregulation and re-regulation elucidated in VBMT stable mixed chimeras within the first 100-day period may represent important mechanisms of tolerance induction.

Animals↗
PubMed1994

Mechanisms of unresponsiveness associated with pretransplant blood transfusion-cyclosporine-induced mixed lymphocyte chimerism.

Multiple pretransplant blood transfusions while under limited daily cyclosporine cover (PTBT-CsA) induce extensive rat renal allograft survival and antigen-specific non-responsiveness. The underlying mechanisms of this extensive allograft survival are not yet fully understood. We hypothesized that one of the potential contributing mechanisms to tolerance induction in PTBT-CsA-treated kidney recipients is the development of stable mixed chimerism, putatively due to the proliferation of stem cells capable of haematopoiesis in the transfused blood. BN rats served as whole blood and kidney donors. LEW rats served as recipients of the PTBT-CsA protocol and BN kidney transplants. Three weekly transfusions were given under concomitant limited CsA cover. Following these multiple primary sensitizations, antigen-specific splenic cellular responsiveness in vivo was normal in comparison with naive animals. However, these experimental splenocytes were non-specifically suppressed against third-party allodeterminants. At 100 days post-transplantation (T100) following tolerance induction to kidney allografts (secondary challenge), in vivo adoptive transfer experiments demonstrated the existence of potent splenic suppressor cells. In vitro suppressor cell assays confirmed that these cells were non-specific suppressor cells. However, following chimerism stabilization at T130, splenic antigen-specific suppressor cells became exclusively expressed in the tolerant animals, replacing the non-specific suppressor cells. At this time, splenic microchimerism was at peak levels and remained stable from T100 to T130. In conclusion, these findings demonstrate that sequential mechanisms of suppressor cell network expression are induced within a chimeric environment by blood-CsA immune modulation. Stable mixed lymphocyte chimerism and related immunomodulatory mechanisms may, therefore, play an important tolerogenic role in blood-CsA-induced non-responsiveness and in the beneficial effect of blood transfusion.

Animals↗
PubMed1992

A rapid and sensitive cellular enzyme-linked immunoabsorbent assay (CELISA) for the detection and quantitation of antibodies against cell surface determinants. I. A comparison of cell fixation and storage techniques.

A solid phase cellular ELISA was designed and evaluated for the detection of antibodies specific for cell surface determinants. It was hypothesized that certain fixation and freezing procedures would result in stabilization of cell structures for prevention of antigen diffusion and extraction during washing procedures. This would assure assay accuracy and convenient sample management. It was hypothesized that fixation with certain reagents prior to analysis would not alter antigenicity of antibody targeted epitopes. In order to improve the preservation of the cells following cell binding to the solid phase matrix while still retaining antigenicity and morphology, a series of fixatives and storage procedures were screened to determine which were best suited for CELISA. Methanol, washing buffer (WB), Hanks' balanced salt solution (HBSS), and 0.5% formalin in HBSS were examined by comparing their relative cell binding capacity and the subsequent cell morphology. In consideration of all variables, fixation in 0.5% formalin provided the best maintenance of cell antigenicity, morphology, binding, and was associated with consistent results. Cells used immediately after fixation and fixed cells used after storage at -80 degrees C for up to 12 months were compared to determine if long term storage affected antigenicity. Since frozen cells and fresh cells demonstrated statistically identical positive to negative ratios and consistency of antibody binding, it was determined that long term frozen storage of formalin-fixed cells did not adversely affect antibody binding capacity to cell surface determinants.

Animals↗
PubMed1991

Vascularized bone marrow transplantation (VBMT): induction of stable mixed T-cell chimerism and transplantation tolerance in unmodified recipients.

In this preliminary report, our model of VBMT across a semiallogeneic barrier consistently brings about antigen-specific host tolerance with absence of GVHD in the majority of recipients. No immunologic or radiologic intervention was utilized. These results emphasized a potentially important mechanism for low-level stable mixed lymphoid chimerism (SMLC) in tolerance induction, independent of immune suppressive effects due to irradiation or immunopharmacologic intervention.

Animals↗
PubMedDate not supplied

A rapid and sensitive cellular enzyme-linked immunoabsorbent assay (CELISA) for the detection and quantitation of antibodies against cell surface determinants. II. Optimal reagent concentrations and predictive analysis.

A cellular enzyme-linked immunosorbent assay (CELISA) was developed for the detection and quantification of antibodies elicited against allogeneic cell surface determinants. The technique uses a solid-phase cell matrix created by fixing cells with a mild formalin solution onto the bottom of a 96-well microtiter plate. A primary layer of alloantisera is first reacted against rat target cells. The secondary antibody, peroxidase conjugated antirat IgG, is then added to each well and serves as the second sandwich layer. Optimal reagent concentrations were determined by serial dilution analysis of various cell concentrations and secondary antibody dilutions. It was found that 200,000 cells per well was the optimal target cell concentration. However, 100,000 cells per well was also sufficient to run the assay with acceptable performance characteristics. Even lower cell concentrations of 10,000 and 20,000 cells/well, although not optimal, also produced acceptable results. Secondary antibody concentration with respect to the optimal cell concentration was determined to be 1:500. At 200,000 cells per well and a 1:500 secondary antibody dilution, the assay presented excellent coefficients of determination and high positive to negative ratios. The reaction was found to be very sensitive in yielding high antibody titers with low background levels and could be defined mathematically as a linear-log function. Titers of multiple unknown alloantibody samples were easily and accurately predicted in an automated manner by regression analysis form known standards. This immunoassay will be useful in studies of cell surface determinant expression and quantitation of antibodies reactive to such markers.

Animals↗
PubMedDate not supplied

Use of regression analysis and flow cytometry for determining levels of mixed semiallogeneic immune chimerism.

It has been shown that tolerance or specific immunologic nonresponsiveness in various lymphohemopoietic transplant models can be associated with the development of mixed lymphoid chimerism. As a specific example, composite tissue (limb) allografts were studied as a model for vascularized bone marrow transplantation (VBMT) and it was demonstrated that development of stable cellular immune chimerism is associated with long-term allograft survival. Recently, studies were initiated using a new parental to hybrid VBMT model, but the detection of donor cells is complicated, due to the fact that they share one parental allotypic determinant. Therefore, regression analysis with a flow cytometric immunofluorescent staining assay was evaluated for the assessment of cellular lymphoid chimerism in donor parental to hybrid (P-->F1) lymphohemopoietic transplant models. Standard curves consisting of known mixed populations of parental donor (Lewis, LEW) and hybrid host F1 (Lew x BN, LBN) lymphocytes were established. Standard curves were analyzed by linear regression statistics and excellent coefficients of determination (r > .881) were obtained for all standard curves. A highly statistically significant (p < .016) linear relationship between level of donor cell chimerism (independent variable) and percent stained (dependent variable) was determined. The technique was then evaluated using the parental to hybrid VBMT model. Levels of donor LEW lymphoid chimerism in all VBMT LBN recipients were successfully assessed by regression analysis and inverse prediction using distinct recipient allodeterminant markers. In conclusion, this technique was proven to be reliable and accurate for the detection of of chimerism in parental to F1 lymphohemopoietic allograft models.

Animals↗
PubMedDate not supplied

Site-specific immunosuppression: mechanisms of cellular immunosuppression that are operative at local and systemic levels.

The cellular mechanisms by which topical cyclosporine A (tCsA) induces site-specific immunosuppression were investigated. Experiments were designed to elucidate how cyclosporine A (CsA) suppresses activated immunocytes in animals that are undergoing local alloactivation and concomitant tCsA immune suppression. Lewis rats received dual Lewis x Brown Norway rat skin allografts; the rats were treated with systemic CsA (sCsA) at 8 mg/kg/day for 10 days after grafting and then tCsA and vehicle thereafter. CsA added to mixed lymphocyte reactions 24 hours after culture initiation modeled the local effects of CsA on alloactivated immunocytes, and tCsA in conjunction with limited sCsA prolonged local skin allograft survival. CsA inhibited both antigen-specific and nonspecific activated alloresponses of immunocytes from animals that had received allografts and that underwent limited sCsA treatment only in a dose-dependent manner. When tCsA had been applied, immunocyte responses to a nonspecific antigen were extremely CsA-resistant as compared with those induced by antigen-specific suppression. However, this nonspecific alloresponse was fully suppressible with the use of elevated CsA doses (66 microg/mL); thus alloresponding immunocytes were significantly more sensitive to CsA if they were challenged with the donor antigen and preexposed to limited sCsA followed by tCsA in vivo.

Administration, Topical↗