Use of the laryngeal mask airway for laser treatment of the subglottis.

A technique for treating subglottic lesions with the intubating laryngeal mask airway is described. It provides unhampered exposure of the subglottis and upper trachea, excellent airway control, and a means of access for ablation with flexible laser bronchoscopy.

Evidence that multiple defects in cell-surface molecule interactions across species differences are responsible for diminished xenogeneic T cell responses.

The purpose of the present study was to identify which of the several possible defects in cell-surface-molecule interactions are responsible for diminished mouse helper T cell responses to xenoantigens. We measured primary mouse anti-monkey, anti-pig, and anti-human proliferation in vitro in experimental systems in which potential defects were partially corrected by lymphokine supplementation and/or the use of transgenic or hybridoma cell populations. We found that the diminished mouse helper T cell responses to xenoantigens result from at least two defects in cell-surface-molecule interactions between T cells and xenogeneic APCs, specifically TCR and/or CD8 interactions with xenogeneic class I MHC molecules and accessory molecule interactions with their ligands (probably LFA-1 with ICAM-1/ICAM-2 and/or LFA-2 with LFA-3). Other investigators have identified additional defects, such as in lymphokine function across species differences. Thus, there appear to be multiple defects responsible for the diminished cellular immune response to xenoantigens.

Xenogeneic proliferation and lymphokine production are dependent on CD4+ helper T cells and self antigen-presenting cells in the mouse.

We studied proliferation and interleukin 2 production by B6 mouse spleen cells in response to stimulation by irradiated cynomolgus monkey spleen cells and compared the results with responses against whole MHC-disparate allogeneic controls (BALB/c). We found that (a) primary xenogeneic helper responses were absent, whereas primary allogeneic responses were brisk, (b) secondary xenogeneic helper responses were dependent on CD4+ T cells and responder antigen-presenting cells (APCs), whereas allogeneic responses could be mediated by either CD4+ or CD8+ T cells independently and were primarily dependent on the presence of stimulator APCs, and (c) secondary xenogeneic helper responses were blocked by an antibody directed against responder class II MHC molecules. These results suggest that mouse helper T cells recognize disparate xenoantigens as processed peptides in association with self class II MHC molecules, similar to the recognition of nominal antigens and unlike direct allo-recognition.

Efficacy of T cell depletion of surgically resected cadaveric marrows by monoclonal antibodies: considerations in HLA-mismatched marrow transplantation.

The potential therapeutic benefits of allogeneic bone marrow transplantation (BMT) remain generally unavailable to patients who have diseases amenable to treatment by allogeneic BMT, but who lack an identifiable HLA-matched marrow donor. If graft versus host disease and graft rejection can be controlled, then the possibility of expanding allogeneic BMT to minimally HLA-matched or fully mismatched combinations exists and a "universal" donor marrow bank might be established. Towards this end, we have evaluated surgical harvest of cadaveric marrow, T cell depletion of such marrow (for prevention of graft-versus-host disease), influence of surgical harvest on final T cell content, and final cell yield. Marrow harvest was coordinated with the donation of other tissues or organs of cadaveric origin. In a series of twenty-two surgical vertebral body harvests, the initial marrow yield per vertebral body was 4.5 x 10(9) with four to six vertebral bodies per harvest. T cell depletion was evaluated by a limiting dilution assay. Since a combination of multiple monoclonal antibodies with specificity for T cell surface molecules provided greater reproducibility in the depletion of T cells from marrow than the utilization of any single antibody, a pool of monoclonal antibodies and complement were used to treat the marrow. The final cell yield per vertebral body was 2.1 x 10(9) for an average total yield of 9.3 x 10(9) cells per harvest. T cell content for each marrow following T cell depletion was less than 0.001%. These marrow have been cryopreserved as a bank of characterized donor marrow for use in HLA minimally matched or unmatched marrow transplantation.

Cardiac allograft survival across major histocompatibility complex barriers in the rhesus monkey following T lymphocyte-depleted autologous marrow transplantation. III. Late allograft rejection.

We have studied organ allograft survival in rhesus monkeys conditioned with myeloablative total-body irradiation and T cell-depleted autologous bone marrow transplantation then given a heterotopic MHC-mismatched cardiac allograft in the immediate postmyeloablative period. This model has enabled us to investigate the role of T cells in vascularized organ allograft rejection. We previously reported (1) that recipients of marrow depleted of T cells below a critical threshold (0.16% residual marrow T cells, or 0.14 x 10(5) infused T cells/kg) experienced a period of freedom from acute rejection associated with a profound nonspecific immune deficiency (determined by skin grafting). Resolution of the nonspecific immune deficiency was associated with late graft rejection. In the present report, we correlate the results of peripheral immune reconstitution studies and direct immunohistochemical analysis with allograft status in order to study T cell subsets involved in late rejection. We report that, in contrast with CD8+/CD28- T cells, CD16+ NK cells, and CD20+ B cells, late allograft rejection was associated with the return of peripheral CD4+ T cells and CD8+/CD28+ T cells, suggesting a critical role for one or both of these subsets in late allograft rejection in this model.

Cardiac allograft survival across major histocompatibility complex barriers in the rhesus monkey following T lymphocyte-depleted autologous marrow transplantation. IV. Immune reconstitution.

Studies of postmyeloablative immune reconstitution have been reported for allogeneic bone marrow transplantation and also for non-T cell-depleted autologous/syngeneic BMT. However, there is a paucity of information regarding immune recovery following T cell-depleted autologous/syngeneic BMT. We have developed a primate transplantation tolerance model in which rhesus monkeys were conditioned with total-body irradiation and extensively T cell-depleted autologous BMT and given a major histocompatibility complex-mismatched heterotopic cardiac allograft. This model provided an opportunity to study peripheral immune recovery following T cell-depleted autologous BMT. Limiting dilution analysis was used to quantify marrow T cells following depletion (2.8% to 25.6% marrow T cells predepletion, 0.00014% to 0.036% residual marrow T cells postdepletion). We found that (1) hematopoietic engraftment was prompt despite extensive marrow T cell depletion, (2) reconstitution of CD4+ helper T cells and CD8+ cytotoxic T cells were substantially delayed (6-12 months) compared with the recovery of CD8+ suppressor T cells, CD16+ NK cells, and CD20+ B cells, (3) distinction between CD8+ cytotoxic T cells and CD8+ suppressor T cells by the CD28 marker was critical in revealing the markedly discrepant recoveries of those subsets, and (4) immune reconstitution resembled that observed in recipients of T cell-depleted allogeneic and non-T cell-depleted autologous/syngeneic BMT, suggesting that the pattern of immune recovery following BMT is not substantially influenced by either allogeneic effects or the number of transferred T cells over a range of values.

Restriction fragment length polymorphism analysis with a cross-reactive HLA class II DR-beta gene probe for the detection of engraftment of MHC-mismatched marrow in the rhesus monkey.

Our interest in MHC-mismatched allogeneic bone marrow transplantation (BMT) in the rhesus monkey prompted us to investigate restriction fragment length polymorphism analysis as a means for detecting lymphohematopoietic chimerism in the primate. A human MHC (HLA) class II DR beta gene cDNA probe was tested on rhesus peripheral blood mononuclear cell DNA digested with any of three restriction enzymes. We found that (1) the human DR beta probe hybridized to as many as 15 restriction fragments per rhesus DNA sample, suggesting cross-hybridization at more than one locus of rhesus MHC class II beta genes; (2) restriction fragment length polymorphisms were common among outbred monkeys as a result of class II beta gene polymorphisms and would be sufficient for chimerism detection in the majority of random pairs of outbred monkeys utilizing only a single restriction enzyme (Bgl II); and (3) sensitivity (5-10% chimerism) was comparable to that reported for restriction fragment length polymorphism assays utilizing non-MHC probes in clinical HLA-identical BMT. Utility of the assay was demonstrated in a preliminary series of experiments in rhesus monkeys conditioned with mixed T cell-depleted MHC-mismatched allogeneic plus T cell-depleted autologous BMT with or without cardiac allograft implantation.

Cardiac allograft survival across major histocompatibility complex barriers in the rhesus monkey following T lymphocyte-depleted autologous marrow transplantation. II. Prolonged allograft survival with extensive marrow T cell depletion.

In the present study, we tested the possibility that a vascularized allograft might induce immunological tolerance in a myeloablated host, similar to the tolerance induced by allogeneic bone marrow grafts. To this end, we developed a rhesus monkey model consisting of myeloablative total-body irradiation and T cell-depleted autologous marrow transplantation followed by MHC-mismatched heterotopic cardiac allograft implantation. Limiting dilution analysis was used to quantify residual marrow T cells following depletion. We found that (1) allograft survival was substantially prolonged in the absence of immunosuppressive drugs (median survival = 160 days) over that seen in controls treated identically but receiving non-T cell-depleted marrow (median survival = 14 days); (2) there was a correlation between allograft survival prolongation and the extent of marrow T cell depletion, with a maximum survival of 329 days associated with a residual marrow T cell content of 0.00014%; (3) nonspecific immune deficiency--and, possibly, specific unresponsiveness of limited duration (determined by cryopreserved donor and third-party skin grafting)--contributed to the rejection-free period seen in recipients of extensively depleted marrow; (4) late allograft rejection occurred in 3 of 3 long-term survivors, thereby demonstrating that permanent tolerance was not induced by the allograft across MHC barriers; and (5) as few as 1.4 x 10(4) infused marrow T cells/kg were sufficient to mediate acute allograft rejection, a threshold approximately 10-fold lower than that reported for the induction of acute graft-versus-host disease following allogeneic bone marrow transplantation.

Experimental cardiac allograft survival across major histocompatibility complex barriers in the rhesus monkey following T lymphocyte-depleted autologous marrow transplantation. I. In vitro T lymphocyte depletion studies.

We have developed a rhesus monkey model consisting of myeloablative total-body irradiation and T lymphocyte-depleted autologous bone marrow transplantation followed by MHC-mismatched heterotopic cardiac allograft implantation that has provided an opportunity to study the role of marrow T cells in cardiac allograft rejection. In order to assess quantitatively the effects of low numbers of residual marrow T cells following depletion, methods to deplete rhesus marrow extensively and to detect residual T cells following depletion at levels below the sensitivity of standard assays have been developed. A rhesus marrow limiting dilution assay has been developed that quantifies less than 1 T cell in 10(5) marrow cells and is superior to traditional detection methods by at least 3 logs. In a direct comparison of four T cell depletion methods, effective depletion has been achieved with complement-mediated cytotoxicity (C'MC), erythrocyte rosetting, and counterflow centrifugal elutriation (CCE), the latter with a simplified single-flow rate protocol. Median marrow T cell depletions of 2.1, 1.1, and 3.1 logs, and total nucleated cell losses of 40%, 61%, and 42% respectively, have been observed. A reported use of ricin A-chain-like toxins for the enhancement of C'MC was of low efficacy with rhesus peripheral blood T cell targets. CCE followed by C'MC has resulted in a median 4.8 logs depletion with residual marrow T cell contents less than 0.001%. Thus, C'MC, E-rosetting, and particularly CCE are effective methods of T cell depletion--and, when used in combination, extensively eliminate marrow T cells. A rhesus marrow limiting dilution assay detects residual T cells at these low levels. These techniques provide a basis for the quantitative study of the role of T cells in organ graft rejection following T lymphocyte-depleted autologous marrow transplantation.

Alternative donor sources in HLA-mismatched marrow transplantation: T cell depletion of surgically resected cadaveric marrow.

Allogeneic bone marrow transplantation has been largely confined in its application to patients with an HLA-matched marrow donor, thereby limiting potential therapeutic benefits for patients with diseases amenable to treatment by marrow transplantation who lack effective alternative therapies and an identified matched marrow donor. T cells in the donor marrow generate graft-versus-host disease which is a major consideration in attempts to widen the application of allogeneic marrow transplantation to the minimally HLA-matched or mismatched setting. The method of marrow harvest influences both the number of such T cells in the donor marrow inoculum and the functional capacity of T cells in murine marrow. We have therefore evaluated surgically resected cadaveric marrow for T cell content and for T cell depletion. Surgically resected marrow is more easily depleted of T cells than aspirated marrow and clinically useful quantities of marrow have been obtained and cryopreserved in a bank of characterized donor marrow for future use in HLA minimally matched or unmatched marrow transplantation.

Modification by the Hancock T6 process of calcification of bioprosthetic cardiac valves implanted in sheep.

The effectiveness of the T6 process (surfactant treatment) to decrease calcification of porcine aortic valvular (PAV) and bovine pericardial (BPV) bioprostheses was investigated. Morphologic and biochemical studies were made of standard and T6-treated PAVs and BPVs that had been implanted for a mean of 20 weeks in the tricuspid position in young sheep. Gross, radiographic, histologic and ultrastructural observations showed that the calcific deposits were less severe in T6-treated (n = 9) than in standard PAVs (n = 7), but were similar in severity in T6-treated (n = 6) and standard BPVs (n = 7). This was confirmed by results of quantitative analyses for calcium in half of each cusp of each explanted valve. Because these results showed large differences in standard deviations in the 4 groups of sheep, natural logarithmic and square-root transformations were used for statistical comparisons. The mean calcium content (milligrams of calcium per gram of dry tissue) of standard PAVs (111 +/- 53) was greater than that of T6-treated PAVs (11 +/- 3) (p = 0.0037). The calcium content of T6-treated PAVs was lower than that of T6-treated BPVs (96 +/- 26) (p = 0.031). However, the calcium content of standard BPVs (35 +/- 13) was not different from that of T6-treated BPVs or standard PAVs. Thus, under conditions of relatively short-term implantation in the sheep model, the T6 process is useful for decreasing the extent of calcification in PAVs, but not in BPVs.

The late prognosis after localized resection for fixed (discrete and tunnel) left ventricular outflow tract obstruction.

We studied the follow-up status of 56 patients after operation for fixed left ventricular outflow tract obstruction (LVOTO), 42 with discrete LVOTO consisting of an obstructing membranous ring in the left ventricular outflow tract (LVOT) and 14 with tunnel (diffuse) LVOTO. Forty-one of the 56 patients were available for long-term follow-up. Patients with discrete LVOTO fared better than patients with tunnel LVOTO postoperatively in their functional class status (discrete: 21 in Class I, five in Class II; tunnel: one in Class I, four in Class II; p less than 0.05), their LVOT peak systolic gradients (discrete: 22 +/- 4 mm Hg; tunnel: 98 +/- 23 mm Hg; p less than 0.02), their actuarially determined survival probabilities (discrete: 82% +/- 9% at 20 years; tunnel: 40% +/- 19% at 20 years; p less than 0.1), and their survival probabilities without an adverse event, i.e., (1) death, (2) reoperation, (3) residual gradient greater than 50 mm Hg, (4) significant aortic regurgitation, (5) bacterial endocarditis, or (6) complete heart block (discrete: 43% +/- 9% at 4 years, 36% +/- 9% at 10 years, and 15% +/- 9% at 20 years; tunnel: 0% at 4 years; p less than 0.02). Thus most patients who undergo operation for fixed LVOTO will survive late postoperatively; resection of the membrane is adequate for relief of LVOTO and for relief of symptoms in most patients with discrete LVOTO; the majority of patients with tunnel LVOTO who undergo only local resection will have an unsatisfactory operative result; most patients with discrete as well as tunnel LVOTO surviving operation will have clinically significant adverse events early or late postoperatively. This last observation dictates continuing long-term follow-up evaluations of patients operated upon for fixed LVOTO.