Eugene E. Emeson

CYCLOSPORIN A IN EXPERIMENTAL LUNG TRANSPLANTATION

Cyclosporin A (Cy A) has been used in combination with low-dose azathioprine (2 mg/kg/day for 14 days) or other immunosuppressives to treat 13 canine lung allograft recipients. Two of five dogs treated with Cy A and azathioprine survive at 13 and 6 months, respectively, with normal lung function and no evident rejection. The other three dogs in this group survived for over 5 months despite evidence of rejection which was reversed with methylprednisolone (50 mg/kg/day for 3 to 5 days). The addition of prophylactic corticosteroids or their substitution for azathioprine resulted in decreased survival without preventing rejection better. The lung allograft rejection that occurred with Cy A was usually later in onset and more easily reversed by corticosteroids than the lung rejection that occurred with standard immunosuppression. Cy A rejection was also sometimes qualitatively different. Perivascular mononuclear cell cuffs and a proportionally greater decrease in allograft perfusion with respect to ventilation were often more prominent than in rejection with standard immunosuppression. In some instances, decreased allograft perfusion evidenced rejection while the plain chest roentgenogram and ventilation remained normal. Except for infection, which only occurred in animals receiving prophylactic corticosteroids, there was no toxicity from Cy A. These findings indicate that this drug is the safest, most effective immunosuppressive agent yet available for use in lung transplantation.

Lectin-dependent cell-mediated cytotoxicity. A new and simple method to quantitate cytotoxic T cell activity in dogs.

Alloimmune T lymphocytes kill a wide spectrum of target cells, even in the absence of alloantigen recognition, providing that a lectin is incorporated into the assay medium. In these studies we have modified the lectin-dependent cell-mediated cytotoxicity (LDCMC) assay to evaluate the cytotoxic activity of canine T cells using a human B lymphoblastoid cell line (Raji) as the target. Canine peripheral blood mononuclear cells activated in vitro in mixed leukocyte cultures or by concan avalin A (Con A) killed 51Cr-labeled Raji target cells (up to 70% in 4 hr) when Con A was included in the assay. T cells expressed cytotoxic activity only when activated and when Con A was present in the assay. Canine T cells activated in vivo also exerted cytotoxic activity in the LDCMC assay. T cells obtained from a rejecting allografted lung by bronchoalveolar lavage displayed considerably higher cytotoxic activity than did T cells obtained from the contralateral normal lung or peripheral blood of the same dog. In contrast, T cells from the lungs and blood of normal dogs and lung-allografted dogs without rejection exerted no cytotoxic activity. We conclude that the LDCMC assay using xenogeneic Raji cells as targets is a valid measure of the cytotoxic activity of canine T cells. Its major advantages include the fact that it is simple to perform and can be used when specific target cells are not available.

Concanavalin A-dependent cell-mediated cytotoxicity in bronchoalveolar lavage fluid. Correlation with lung allograft rejection in mongrel dogs during cyclosporine dose tapering.

Although cyclosporine (CsA) is widely used as the primary agent for inhibiting the rejection of organ allografts in man, the ideal immunosuppressive regimen for utilizing this drug is still uncertain. To investigate this question, a concanavalin A (con A)-dependent cell-mediated cytotoxicity (CDCMC) assay was used to examine the development of intragraft and peripheral blood cytolytic T lymphocyte activity during CsA dose tapering. These studies were conducted in a canine single-lung transplantation model that facilitates serial examination of intragraft effector cells by bronchoalveolar lavage (BAL). A remarkable correlation of increased intragraft CDCMC and clinical evidence of lung allograft rejection was observed during CsA dose tapering in some recipients. In other recipients CDCMC remained low and evidence of rejection was not observed during drug tapering. In contrast, peripheral blood CDCMC did not correlate well with evidence of rejection. Rejection phenomena observed after termination of CsA therapy were reversed by resumption of CsA treatment but were not reversed by administration of methylprednisolone. Furthermore, the increased level of CDCMC was diminished by reinstitution of CsA therapy at the initial dosage. Following termination of CsA therapy, a prolonged period of unresponsiveness was observed in nearly two-thirds of the recipients, and 60% of these latter dogs had unlimited survival of their lung allografts (median greater than 496 days). Intragraft CDCMC remained low during the periods of unresponsiveness and increased upon onset of rejection. We conclude that measurement of intragraft CDCMC is a useful in vitro method of monitoring lung allograft rejection, and therefore provides a technique for adjusting CsA dosage schedules to achieve maximally effective immunosuppression. The use of this assay for monitoring rejection of other organ grafts requires further investigation.

Bronchial anastomotic healing in canine lung allotransplants treated with cyclosporine

Bronchial anastomotic healing was evaluated in 22 long-term-surviving canine lung allotransplant recipients treated with cyclosporine as the major immunosuppressive agent. Mean survival in these dogs was over 155 days, and 4 animals survived 1-3 years. Bronchial anastomotic complications were limited to 5 cases of minimal (less than 15%) bronchostenosis. The bronchial anastomoses became somewhat edematous and friable during rejection episodes, but no clinically serious sequelae--such as hemorrhage, peribronchial abscess, or bronchial dehiscence--were observed. Gross and microscopic evaluation of the recipient and donor segments of the anastomoses revealed excellent healing, with only scattered areas of inflammatory cells. The decreased frequency and severity of rejection episodes in animals treated with cyclosporine permits early revascularization of the bronchus to take place and reduces the need for other immunosuppressive agents that may interfere with bronchial healing. Cyclosporine is an effective immunosuppressive agent for canine lung allotransplantation and allows normal bronchial anastomotic healing to occur.

Cyclosporin A as the initial immunosuppressive agent for canine lung transplantation: Short-and long-term assessment of rejection phenomena

Cyclosporin A (Cy A) was evaluated as the initial immunosuppressive agent for lung transplantation in unmatched mongrel dogs. Rejection phenomena were assessed by plain chest roentgenograms, histology, radionuclide ventilation-perfusion scans, and by the level of lectin-mediated lymphocyte cytotoxicity in cells obtained by bronchoalveolar lavage. Two groups of lung allograft recipients were studied. One group was treated with Cy A alone and the other was treated with Cy A and a short course of azathioprine (AZA) therapy (2 mg/ kg/day for 14 days). Recipient survival and the nature and severity of rejection phenomena were similar in the two groups. Two morphologically and functionally distinct patterns of rejection occurred with similar frequency in Cy A and Cy A-AZA-treated lung allograft recipients. The first pattern was characterized by extensive changes in plain chest roentgenograms, the presence of interstitial, perivascular, and alveolar mononuclear cell infiltrates in histological sections, both ventilation and perfusion abnormalities on lung scans, and relatively high levels of lectin-mediated T cell cytotoxicity. The second pattern of rejection was characterized by minimal changes on chest films, histological findings limited to perivascular mononuclear cell infiltrates, relatively normal ventilation but abnormal perfusion on lung scans, and significantly lower T cell cytotoxicity. High-dose, short-term treatment with methylprednisolone was usually effective in the reversal of the first type of rejection. In contrast, long-term maintenance prednisone therapy that was begun >30 days after transplantation was necessary to suppress the second pattern of rejection. Our study suggests that Cy A alone provides effective initial immunosuppression for use in lung allotransplantation, although corticosteroid therapy may be required after the 1st month. Preliminary data suggest that it may be possible to discontinue Cy A therapy without evidence of increased lung allograft rejection in some mongrel dogs.

Identification of canine t lymphocytes with antidog lymphocyte serum and antirat lymphocyte serum by indirect immunofluorescent staining.

Reliable methods for the identification of T lymphocytes in the dog have not been available. We determined the proportion of T cells in canine lymphoid tissues by an indirect immuno-fluorescent assay utilizing rabbit anti-rat thymocyte (RART) serum. We found that RART, gave values similar to those of rabbit anti-dog lymphocyte (RADL) serum. RADL, however, required absorption before it was T cell-specific while absorption was unnecessary with RART. Both heterologous antisera stained greater than 90% of the dog thymocytes, 60% of the lymph node cells, 40% of the spleen and peripheral blood cells, and only 5% to 10% of the bone marrow cells. The specificity of RART was further demonstrated by its high reactivity with enriched canine T cell subpopulations obtained from nylon-wool columns, preparative EAC rosette formation, and preparative cell electrophoresis. In contrast, lymphocyte populations enriched for B cells in the same experiments exhibited low reactivity with RART. Also, RART gave low reactivity with B cells obtained from dogs with lymphoproliferative diseases (<90% positive for surface IgG). A double labeling immunofluorescent technique demonstrated no overlap (<1%) between RART-positive cells and cells with surface IgG. These studies indicate that RART is a convenient and reliable reagent for the enumeration of canine T lymphocytes. This methodology should be useful for assessment of immune function in the dog.