Irreversible chronic vascular rejection occurs only after development of advanced allograft vasculopathy: a comparative study of a rat cardiac allograft model using a retransplantation protocol.

Indefinitely surviving WF.1L (RT1(1)) cardiac allografts transplanted to LEW (RT1(1)) recipients provide an ideal model for controlled comparative studies of chronic vascular rejection (CVR). To determine the stage of development at which the progressive CVR can be reversed when deprived of an ongoing recipient alloimmune response, WF.1L-LEW cardiac allografts were retransplanted back into syngeneic donor strain WF.1L recipients at specific time periods after initial allogeneic engraftment and were maintained in WF.1L syngeneic hosts for a further 40 days. The vascular changes in the retransplanted allografts were compared with those of nonretransplanted allografts and with nonretransplanted and retransplanted LEW-LEW isografts examined at similar time periods. The early vasculopathic inflammatory changes were consistently reversed by retransplantation of the cardiac allografts back into syngeneic recipients after 20 days and 40 days of allotransplantation. Syngeneic retransplantation of the cardiac allografts at 60 days after allotransplantation did not reverse the essentially nonvasculitic occlusive vasculopathy invariably present in WF.1L-LEW cardiac allografts at this time period. Thus, the vasculitic and minimal subocclusive myointimal changes associated with early CVR in this model are alloantigen dependent and reversible. Irreversible CVR occurs only after advanced proliferative vasculopathy has been established in the allogeneic host.

Increased endothelin expression in a rat cardiac allograft model of chronic vascular rejection.

Endothelins (ET) are potent vasoconstrictors that are directly mitogenic for vascular smooth muscle cells and fibroblasts. It is possible that the vasoconstrictor and mitogenic effects of ET could play a significant role in the vascular remodeling process that occurs in chronic vascular rejection (CVR). We have previously shown that cardiac allografts in the indefinitely surviving major histocompatibility complex identical WF.1L (RT1(1)) to Lewis (LEW) (RT1(1)) inbred rat strain combination provide a highly reproducible model of progressive CVR. The objective of this investigation was to measure endothelin-1 ventricular content of WF.1L-LEW cardiac allografts and to determine the immunohistochemical patterns of ET cellular reactivity at well defined posttransplant time periods. Data were compared with those obtained in similar studies of LEW-LEW syngeneic: heart grafts as well as all recipients' own hearts. The ventricular ET-1 content of the WF.1L cardiac allografts was markedly higher (4.3-, 7.0-, and 4.8-fold at 20, 40, and 60 days, respectively) than in corresponding recipients' hearts. Also, the increase in ventricular ET-1 levels as compared with the recipients' hearts rose significantly only in the allograft group. No comparable differences were observed in the syngeneic heart graft controls. Allografts consistently showed ET staining of intimal myocytes at sites of occlusive and subocclusive intimal proliferation associated with CVR. Allografts also showed ET cellular staining in areas of reparative fibrosis associated with indolent interstitial rejection and ischemic myocardial damage. The results of this study strongly suggest that ET may play a significant role in the pathogenesis of CVR.

Evidence that recipient CD8+ T cell depletion does not alter development of chronic vascular rejection in a rat heart allograft model.

Progressive chronic vascular rejection is a central feature of indefinitely surviving WF.1L LEW/Gut (RT1(1)) heart grafts transplanted to LEW (RT1(1)) recipients in unmodified donor-recipient combinations. At 70 days posttransplantation, large vessels of the grafts are characterized by the presence of vasculitis, vasculitis with associated variable myointimal thickening, and occlusive myointimal thickening with minimal or absent concomitant vasculitis. To assess the potential role of CD8+ T cells as critical effectors of chronic vascular rejection in this model, LEW recipients of WF.1L heart grafts were effectively depleted of CD8+ T cells as a result of prior thymectomy and anti-CD8 (MRC OX8) monoclonal antibody administration prior to transplantation. WF.1L heart grafts transplanted to LEW recipients that had undergone prior sham thymectomy and MRC OX8 administration, or thymectomy and administration of antibody-free culture supernatant, provided appropriate controls. At 70 days posttransplantation, large vessels of WF.1L heart grafts in all 3 transplantation groups showed similar morphologic features, which were comparable to those observed in heart grafts of long-surviving unmodified donor-recipient pairs. This study has shown that profound selective depression of recipient CD8+ T cells does not alter the characteristic features of chronic vascular rejection in this rat cardiac model, and provides evidence that CD8+ T cells play no critical role in the initiation or development of progressive vascular damage in this setting.

Multiple patterns of MHC class II antigen expression on cellular constituents of rat heart grafts. Lack of correlation with graft survival, but strong correlation with vasculitis.

The patterns of induced major histocompatibility antigen expression on indigenous cellular elements of heterotopic rat cardiac grafts were determined by immunohistologic methods in a variety of donor-recipient combinations. Heart grafts were studied in combined full-MHC- and non-MHC-disparate combinations, isolated intra-MHC-disparate combinations, and non-MHC-disparate combinations. The pattern of class II expression on cellular constituents of the grafts was highly variable and critically dependent upon the nature of the specific unidirectional donor-recipient combination. No uniform pattern of class II expression emerged that was clearly predictive of rapidity of rejection or of protracted survival. However, vasculitis was confined to grafts in combinations in which induced class II expression on graft large vessel endothelium was present. Sites of vasculitis were never encountered in the absence of induced class II expression on overlying endothelium. Vasculitis and associated induced class II expression on large vessel endothelium were present in rapidly rejecting grafts and in grafts with indefinite survival. In the latter, vasculitis was shown to progress to a late phase of occlusive intimal thickening. Induced class I expression on graft cardiac myofibers was present in all the genetically disparate donor-recipient combinations examined in this study, irrespective of the length of graft survival. This investigation has shown that no uniform stereotyped pattern of MHC antigen expression on cellular constituents correlates with the length of graft survival. However, induced class II expression on graft large vessel endothelium is closely associated with vasculitis, which can directly progress to occlusive intimal thickening in grafts with prolonged survival.

Morphologic studies of acute rat cardiac allograft rejection across an isolated major histocompatibility complex class I (RT1A) disparity.

To define the morphologic correlates of acute rat cardiac allograft rejection across an isolated major histocompatibility complex (MHC) class I disparity, rejecting PVG.R1 cardiac allografts transplanted to (PVG x WF)F1 recipients were studied from days 4-8 posttransplantation. Documented ultrastructural tracer techniques as well as immunohistologic and immunoelectron microscopic methods were employed for morphologic analysis. Using intravenously administered horseradish peroxidase as a tracer probe for cell membrane permeability dysfunction, it was shown that severe diffuse loss of integrity of the microvascular endothelium preceded functional rejection, providing strong evidence that the allograft microcirculation is a central target of graft destruction. Also, rejection was associated with localized cardiac myofiber alterations prior to development of significant endothelial changes, indicating that cardiac muscle cells are additional cellular targets of immunologic injury. The ultrastructural features of progressive endothelial and myofiber injury, the predominance of MRC OX8+ lymphocytes and MRC OX6+ macrophages sequestered within the grafts, and the pattern of donor class I expression by allograft endothelium and cardiac myofibers were similar to those observed in rejecting allografts in full MHC-disparate combinations. Since it has been previously shown that MRC OX8+ class I-reactive T cells are absolutely required for rejection in this isolated class I-disparate model, the morphologic data raise the possibility that the OX8+ T lymphocyte subpopulation may also play a highly significant role in rat cardiac allograft rejection across a full MHC disparity.

Dendritic cell-lymphoid cell aggregation and major histocompatibility antigen expression during rat cardiac allograft rejection.

To determine the pattern of cellular expression of donor MHC class I and class II antigens during the course of rat cardiac allograft rejection, ACI cardiac allografts transplanted to BN recipients were examined from day 2 to day 6 using immunohistologic and immunoelectron microscopic methods. We used both monomorphic and donor-specific mouse anti-rat MHC class I and class II mAbs in this study. In normal ACI hearts, MHC class I reactivity was confined to the vascular endothelium and to interstitial cells. Ongoing rejection was characterized by an increased donor MHC class I staining intensity of microvascular endothelium and induction of donor class I surface reactivity on cardiac myofibers. Donor MHC class II reactivity was exclusively confined to interstitial dendritic cells (IDC) in both normal ACI hearts and in rejecting allografts, although rejection was associated with marked fluctuations in class II IDC frequency. An early numerical depression in class II IDC present in both allografts and syngeneic heart grafts was attributed to a direct effect of the transplantation procedure. By days 3-4, allografts showed an absolute overall increase in donor class II IDC frequency, which was associated with the presence of multiple localized high-density IDC-lymphocyte aggregates. The lymphocytes present in the focal areas were predominantly of the class II-reactive Th cell subpopulation. These aggregates may thus represent the in vivo homologue of dendritic cell-lymphocyte clustering, which has been shown to be required for primary class II allosensitization in the rat and mouse in vitro. During the late phase of rejection, there was a marked numerical fall in donor class II IDC, which correlated with extensive overall graft destruction. This study has shown that acute rat cardiac allograft rejection can occur in the absence of donor MHC class II expression by allograft vascular endothelium and cardiac myofibers. The IDC, which are believed to represent the principal class II alloantigen presenting cells in the rat heart, remain the sole class II-expressing cellular constituents of the graft throughout the course of rejection.

Comparative immunohistologic studies in an adoptive transfer model of acute rat cardiac allograft rejection.

It has been shown that fulminant acute rejection of rat cardiac allografts across a full haplotype disparity may occur as a direct result of adoptive transfer of sensitized W3/25+ MRC OX8- SIg- T helper/DTH syngeneic spleen cells to sublethally irradiated recipients. In order to establish the immunohistologic parameters of this form of rejection, allografts and recipient lymphoid tissue were analyzed using a panel of monoclonal antibodies of known cellular distribution. These data were compared with those obtained following reconstitution of irradiated allograft recipients with unseparated sensitized spleen cells, with unreconstituted irradiated donor recipient pairs, with unmodified first-set rejection, and with induced myocardial infarction of syngeneic heart grafts transplanted to normal and to sublethally irradiated recipients. Rejecting cardiac allografts transplanted to all reconstituted irradiated recipients were characterized by extensive infiltration with MRC OX8+ (T cytotoxic-suppressor, natural killer) cells even when this subset was virtually excluded from the reconstituting inocula. A similar proportional accumulation of MRC OX8+ cells observed at the infarct margins of syngeneic heart grafts transplanted to irradiated unreconstituted recipients greatly exceeded that present in normal nonirradiated controls. These data provide evidence that under conditions of heavy recipient irradiation, MRC OX8+ cells may be sequestered within heart grafts in response to nonspecific injury unrelated to the rejection process. Although there was no significant degree of MRC OX8+ cellular repopulation within organized secondary lymphoid tissues of irradiated animals over the study period, the density of ileal mucosal MRC OX8+ lymphocytes approximated normal at 7 days post-irradiation, raising the possibility that these cells could share a common origin with those sequestered within the heart grafts. Carbon+ MRC OX6+ macrophages were a significant component of the infiltrate in all rejecting cardiac allografts, as well as in all infarcted syngeneic heart grafts--providing further evidence that macrophage "activation" with expression of class II determinants may occur in response to nonspecific injury. In unmodified first-set rejection there was an intense B cell reaction in recipient spleens and lymph nodes. In the adoptive transfer model, marked B cell expansion was exclusively confined to the parathymic lymph nodes of irradiated allograft recipients reconstituted with the sensitized W3/25+, MRC OX8-, SIg- T helper/DTH donor cell inocula.(ABSTRACT TRUNCATED AT 400 WORDS)

Leukocyte subsets in first-set rat cardiac allograft rejection. A serial immunohistologic study using monoclonal antibodies.

For an immunohistochemical analysis of the cells infiltrating rat cardiac allografts undergoing unmodified first-set rejection, functioning Wistar Furth (RT1u) cardiac allografts transplanted to Lewis (RT1(1)) recipients were arbitrarily removed at days 3-6 posttransplantation, and were evaluated histologically using a series of monoclonal antibodies (MAbs) of known cellular distribution, the binding of which was visualized by an indirect immunoperoxidase technique. Sequestered cells present in cryostat sections were segregated numerically on the basis of staining for MAbs detecting T cells, some NK cells, and neutrophils (W3/13); cytotoxic suppressor T cells and most NK cells (MRC OX8); helper T cells and all macrophages (W3/25); B cells and a macrophage subpopulation (MRC OX6, Ia common part determinant); immature T and B cells (MRC OX7, Thy 1.1); and all leukocytes (MRC OX1, LCA). The proportion of cells stained for each MAb was expressed as a percentage of all estimated leukocytes as determined directly on the hematoxylin counterstained sections. Macrophages expressing the W3/25 and MRC OX6 antigens were enumerated on the basis of intracytoplasmic accumulation of previously administered colloidal carbon. There was a significant fall in the relative proportion of W3/25+ carbon- cells within allografts as a function of time. The proportion of mononuclear cells staining for W3/13 and for MRC OX8 showed no statistically significant variation during the period of study. There was a correlation at all periods between the proportional sum of W3/25+ carbon- cells and MRC OX8+ cells and the proportion of W3/13+ mononuclear cells present. The W3/25+ carbon-:MRC OX8+ ratio showed a significant linear decrease with duration of allograft placement. The proportion of carbon+ cells expressing the MRC OX6 and the W3/25 antigens showed a highly significant rise as a function of time. Nonspecific esterase-stained cells showed a similar sequential pattern. These data provide well defined immunohistologic parameters of leukocyte subsets present in unmodified first-set cardiac allograft rejection in the rat.

Cystolytic events and the possible role of germinal cells in metastasis in chronic alveolar hydatidosis.

Two and six week old alveolar cysts of Echinococcus multilocularis were isolated from C57BL/6J and Balb/cJ mice and used to study the cystolytic events mediated by resident inflammatory cells. Ultramicroscopic degenerative changes in these cysts were compared with the ultrastructure of in vitro grown cysts (free of inflammatory cells). Macrophages, eosinophils and neutrophils were found to bind to the laminated layer of plasma membrane of the germinal layer of cysts. Hydrolytic enzymes and eosinophil granules released extracellularly from inflammatory cells at the cyst surface appear to dissolve the laminated layer and produce gaps in the plasma membrane. Macrophages actively phagocytose the granular ground substance of the disintegrated laminated layer. Cystolysis results in the release of germinal cells which measure 6-17 microns, possess nuclei with prominent nucleoli, sparse endoplasmic reticulum, Golgi complexes and oval mitochondria. Based on histologic evidence we believe that germinal cells may be the progenitors of cysts. The possibility that germinal cells may give rise to distant metastatic foci of alveolar cysts, in immunocompromised hosts, is discussed.

A controlled serial ultrastructural tracer study of first-set cardiac allograft rejection in the rat. Evidence that the microvascular endothelium is the primary target of graft destruction.

In order to define the serial morphologic correlates of unmodified first-set cardiac allograft rejection in an inbred rat strain combination, a series of Wistar-Furth cardiac allografts transplanted to normal nonsensitized Lewis recipients were studied as a function of time with the use of well-documented ultrastructural tracer techniques. Colloidial carbon was employed as a vascular label for detection of microvascular endothelial structural alterations, and horseradish peroxidase was used as a tracer probe for localization of cell-membrane permeability dysfunction of allograft endothelium and cardiocytes as well as of elements of the cellular infiltrate. Wistar-Furth to Wistar-Furth syngeneic heart grafts and Wistar-Furth recipients' own hearts provided appropriate control data. This study was demonstrated that severe diffuse loss of functional and structural integrity of the microvascular endothelium precedes the development of extensive damage to cardiac muscle cells and thus provides strong evidence that the allograft microcirculation is the primary target of immunologic injury. In addition, the sequential pattern of injurious changes present in the rejecting allografts was similar to that observed in certain models of delayed-type hypersensitivity and of skin graft rejection, raising the possibility that lymphokine-mediated mechanisms may be of major pathogenetic significance in this setting.

Effect of preparatory techniques on the gross morphology of Mycoplasma hominis.

A method is presented whereby cells of Mycoplasma hominis can be prepared with minimal distortion for electron microscopy. After the addition of glutaraldehyde to broth cultures, incubation is continued for 1 h. The cells are then collected by centrifugation, washed in distilled water, and used for negative-contrast preparations.

Mycoplasma hominis: growth, reproduction, and isolation of small viable cells.

Improved methods for studying the growth of Mycoplasma hominis (ATCC 14027) have been developed, involving modified growth conditions and preparation of the organisms under minimally distorting conditions. Cells so prepared from batch cultures show relatively uniform exponential growth and appear to be dividing by binary fission; but pleomorphic forms appear upon further incubation. Similar behavior was demonstrated by another laboratory-adapted strain and by three clinical isolates, and therefore seems characteristic of the species. The pleomorphic populations contain small forms having diameters within the 100- to 250-nm size range reported for "elementary bodies." Such forms were isolated from this strain of M. hominis by sequential filtration using gravity alone, after cell aggregates were dispersed by Pronase treatment. Of the small bodies which traversed membranes of 220-nm pore size, a negligible number grew in liquid or on solid media, suggesting that these were not essential reproductive units in a life cycle, but involution forms due to growth in an altered environment.