Muscarinic receptor subtypes in the porcine lung during postnatal development.

The responsiveness of the pulmonary circulation to acetylcholine changes in the newborn piglet. Therefore muscarinic receptors have been studied in the developing porcine lung from birth to adulthood using ligand binding, Northern blotting and in situ hybridisation. Maximal binding capacity of [N-methyl-3H] scopolamine and the affinity of the receptor in lung membranes increased between birth and 16 days (p < 0.05). Subtype affinity changed with age, but always M3, > M1 > M2. Northern blots of porcine muscarinic receptor subtypes showed m1, m2 and m3 mRNA present in lung membranes. m2 mRNA was present at all ages and decreased with age. m1 mRNA was absent at birth and m3 mRNA was absent at 3 days. Autoradiographic localisation showed ligand binding to the parenchyma and airway smooth muscle and nerves, there was no binding to intrapulmonary vessels. In situ hybridisation localised mRNA of all three subtypes to the smooth muscle cells of both vessels and airways. Changes in the receptor subtypes may explain the pharmacological changes during postnatal adaptation.

Anti-neutrophil cytoplasmic antibodies and anti-endothelial cell antibodies are not increased in Kawasaki disease.

We studied anti-neutrophil cytoplasmic antibodies (ANCA) and anti-endothelial cell antibodies (AECA) in 58 children with acute Kawasaki disease (KD) before i.v. gamma globulin treatment, 35 children with infection and fever > 38.5 degrees C, and 48 healthy afebrile children. ANCA were studied by indirect immunofluorescence (IIF) on ethanol-fixed neutrophils and by ELISA with crude neutrophil extract as antigen. AECA were studied using ELISA on resting and activated endothelial cells. ANCA IIF was weakly positive, cytoplasmic, diffuse and homogeneous in all three groups. ANCA IIF, ANCA ELISA and AECA ELISA were no higher in KD than in febrile children. There was no difference between KD with and KD without coronary artery aneurysms. AECA differences between the KD and afebrile group were not significant after correction for total IgM. In contrast with our previous findings, we conclude that ANCA and AECA are not raised in KD compared with febrile controls. It therefore seems unlikely that they are important in the pathogenesis of vasculitis in KD.

Noninvasive monitoring of human cardiac allograft rejection.

The frequency of donor-reactive cytolytic T lymphocytes was measured in the peripheral blood mononuclear cell population of a group of 12 cardiac allograft recipients immediately before and at various time points after transplantation. At each of the time points after transplantation the donor heart was biopsied and the rejection status of the graft was determined by applying standard histological criteria. The results of this study showed that the preoperative frequency of donor-reactive cytolytic T lymphocytes in the blood was not predictive of a future tendency toward graft rejection. However, when all the data were examined it was apparent that the frequency of donor-reactive cytolytic T lymphocytes was significantly higher (P less than 0.05) in blood samples from patients whose simultaneous biopsy showed histological evidence of acute cardiac allograft rejection than in blood samples from transplant patients showing no evidence of rejection.

Lung transplantation in the rat: a model for study of the cellular mechanisms of allograft rejection.

Single-lung transplantation in the rat has been shown to provide an effective model for the study of cellular events associated with allograft rejection. It is possible to recover sufficient viable immune cells for functional immunological studies by lavage of the broncho-alveolar space of the grafts with tissue-culture medium. These cells are representative of the population within the parenchymal infiltrate, but are not exposed to harsh, potentially damaging, physical and chemical conditions during their extraction. Lavage-derived cells from non-immunosuppressed recipients showed donor-specific cytotoxicity, were clonable by limiting dilution culture, and proliferated in response to 24-hr stimulation with recombinant IL-2. Administration of Cyclosporin A (CsA) prevented pulmonary rejection and was also shown to block the formation of specific cytotoxic effector cells and the development of responsiveness to IL-2.

Rat heterotopic heart transplantation: quantification and analysis of cell mediated cytotoxicity.

Limiting dilution analysis was used to measure the frequency of PVG-reactive cytotoxic T lymphocytes (CTL) within the peripheral blood mononuclear cell population of Lewis rats before heterotopic transplantation of PVG rat derived cardiac tissue, and in both the blood and graft-infiltrating cell populations at daily time points afterwards. Before surgery, the frequency of PVG-reactive cells within the blood was between 1/31,700 and 1/50,300; however, this value increased rapidly on day 4 after transplantation to reach values of up to 1/1,100 by day 7. The frequency of these cells was first measurable in the graft-infiltrate on day 2 and also showed a rapid increase 4 days after surgery; peak values up to 1/4,800 were recorded on day 5. This time corresponded with that of functional cardiac rejection and maximum infiltration of the graft by mononuclear cells. The similar kinetic changes and absolute values recorded for the frequency of donor-reactive CTL within the blood and graft-derived cell populations was indicative of a rapid bi-directional passage of cells between these pools and provided no evidence for specific sequestration of CTL by the graft. Cells purified from the graft on post-operative day 5 mediated an immediate specific cytotoxicity towards PVG target cells (44% lysis during a 4 h assay at an effector:target ratio of 100:1) which was of a higher activity than would be predicted on the basis of an effector population containing only 1/4,800 PVG-reactive CTL. This finding implies that other mononuclear cell types than CTL present within the graft-infiltrating population were capable of mediating target cells lysis.

Canine unilateral lung transplantation: effect of ciclosporin A on the frequency of donor-lytic lymphocytes.

Limiting dilution analysis showed that the frequency of precursor donor-lytic cytolytic lymphocytes increased dramatically within the leucocyte population recovered by broncho-alveolar lavage in dogs after unilateral lung transplantation. The increases for animals experiencing acute pulmonary rejection (11.5-24.8 times pre-operative level) and for those receiving long-term ciclosporin A therapy (5.4-17.6 times pre-operative level) were similar. Therefore, it appears that ciclosporin A does not prevent the sequestration of precursor donor-lytic cytotoxic cells within lung allograft tissue.

Precursor frequency of donor-specific lymphocytes recovered from canine lung transplants.

Dogs receiving left unilateral allo- or auto-grafts were treated with Cyclosporin A (Cy A) for 4 days. Thereafter allografted transplant recipients showed pulmonary pathology consistent with rejection. Blood and bronchoalveolar lavage (BAL)-derived lymphocytes were isolated from the recipient animals before and at various times after operation and the frequency of allospecific precursor cytolytic lymphocytes (pCTL) determined by limiting dilution analysis (LDA). Samples from the autografted control animal did not show any post-operative frequency changes; however, both blood and BAL-derived lymphocytes from allografted recipients showed a significant post-operative increase in the proportion of donor-specific pCTL. This increase was consistently greater in samples from the transplanted than the autochthonous lung. The frequency of pCTL determined using targets from an unrelated dog showed no post-operative increase. It is likely that the increase in frequency of donor-specific pCTL recorded during graft-rejection is a specific consequence of an interaction between the graft and recipient's immune system.

Lung transplantation: effect of cyclosporine A on the frequency of cytolytic lymphocytes recovered from blood and the bronchoalveolar space.

The frequency of donor-specific cytolytic cells within the inflammatory infiltrate produced in the graft after canine unilateral lung transplantation was measured using a limiting dilution assay. Two groups of recipient animals were studied; animals in the first group were treated with oral cyclosporine A for 4 days after transplantation, after which the drug was withdrawn thereby allowing acute rejection to begin as evidenced by histologic examination; animals in the second group were treated with cyclosporine A for the duration of the experiment and developed a subacute, alveolar manifestation of rejection. Frequency analysis was performed for lymphocytes isolated from the peripheral blood and, using the technique of bronchoalveolar lavage, lung allograft for each recipient animal both before transplantation and at various times after the operation. The group of recipients that was treated with cyclosporine A for 4 days showed an increase in the frequency of donor-specific cytolytic cells in the blood and lavage-derived lymphocyte populations that reached peak values (four to eight and five to eighteen times the preoperative level, respectively) between days 9 and 14 after operation. The frequency of these cells remained constant at a preoperative level into the peripheral blood of recipient animals treated long term with cyclosporine A; however, the frequency within the graft-derived lymphocyte population increased to reach peak values (12 to 25 times the preoperative level) between days 5 and 15 after operation. In conclusion, these results show that although cyclosporine A modifies pulmonary rejection pathologic conditions, this drug does not prevent the sequestration of donor-specific cytolytic cells within the allograft.