Chimeric pig hearts resist hyperacute rejection in ex vivo perfusion model.

With surrogate tolerogenesis. the recipient immune system is engrafted within the donor pig before organ transplant. Chimeric pig hearts may resist hyperacute rejection by inducing accommodation. This hypothesis was tested using an ex vivo isolated piglet heart perfusion model. Processed sheep marrow was infused into fetal pigs at 45 days gestation. Heart explants from chimeric or nonchimeric pigs were suspended in a Langendorff apparatus and perfused with plasma from unsensitized sheep or sensitized sheep. Nonchimeric hearts perfused with plasma from unsensitized functioned for 240 min (N = 3). Nonchimeric hearts perfused with sensitized plasma deteriorated rapidly, functioning at 19+/-12 min (N = 6); Immunohistochemistry of heart graft revealed extensive deposition of IgG, IgM in the microvascular. In contrast, chimeric hearts perfused with sensitized plasma functioned for 183+/-46 min (N = 3)(p <.001); Deposition of IgG, IgM had substantially less. Heart grafts procured from chimeric pigs survived in the presence of antidonor IgG, IgM, and complement, demonstrating that chimeric pig hearts resist hyperacute rejection.

Effect of surrogate tolerogenesis on the vascular rejection of pig heart xenografts.

BACKGROUND: Organ xenografts are fulminantly rejected by antibody-mediated vascular rejection. Surrogate tolerogenesis (ST), the induction of tolerance within the donor, is effective with aorta xenografts. This preliminary study assesses the effect of ST on preformed antibodies and rejection of porcine heart xenografts. METHODS: Tolerance to the donor pig was induced by infusing recipient marrow into fetal pigs. Later, pig splenocytes were transfused and heterotopic pig hearts transplanted using chimeric or nonchimeric pigs. Anti-pig antibodies were assessed. RESULTS: With ST alone, xenografts developed cellular rejection at 4-6 days, whereas control grafts developed vascular rejection at 3-4 days (cellular vs. vascular, P<0.03). There was a reduction in preformed antibodies (P<0.03). ST combined with moderate cyclosporine prevented rejection at 9+ and 25 days in sensitized recipients compared with vascular rejection at 0.5-2 days for controls (P<0.07). CONCLUSIONS: ST seems to provide protection against vascular rejection. The cellular rejection seems sensitive to cyclosporine.

Denuded bowel after recovery from graft-versus-host disease.

Graft-versus-host disease (GVHD) is a common complication of bone marrow transplantation and often involves the gastrointestinal system. It is unclear whether there can be severe enough damage by GVHD to permanently injure the bowel and thereby prevent mucosal regeneration. We describe a patient who had successful treatment of GVHD, but who had such severe scarring of the bowel mucosa that the colonic epithelium could not regenerate even 50 days after biopsy-demonstrated resolution of GVHD. Surveillance cultures and histological analysis indicate that this denudation was not caused by infection or continued GVHD. This is an important observation with implications for monitoring response to GVHD therapy and using rectal biopsy to evaluate GVHD.

Recruitment of semiallogeneic dendritic cells to the thymus during post-cyclosporine thymic regeneration.

Based on studies of the thymic microenvironment in the model of cyclosporine (CsA)-associated syngeneic graft-versus-host disease, we have hypothesized that immune tolerance develops after CsA is stopped, as the thymus regenerates and recruits new dendritic cells. CsA normally induces medullary involution with destruction of the medullary dendritic cells (DC) and epithelium. This principle could provide practical advantages for transplantation if it is used to recruit new DC into the thymic medulla. Here we administer LEW x BN F1 splenocytes to BN rats and treat them with a short course of CsA. After CsA is stopped, the F1 cells are rapidly recruited to the thymus, and by 10 days after CsA, they are localized at the corticomedullary junction, the natural location of thymic DC. In contrast, dexamethasone, which induces cortical involution, did not lead to thymic recruitment of F1 DC. Recruitment was better if the splenocytes were administered before CsA was stopped. Engraftment of the thymus was also achieved using bone marrow and temporary skin grafts as sources of DC. These observations provide the basis for a novel approach to inducing tolerance to alloantigens with minimal immune suppression and define the goals for further development.

Characterization of the autoreactive T cell repertoire in cyclosporin-induced syngeneic graft-versus-host disease. A highly conserved repertoire mediates autoaggression.

Syngeneic graft-vs-host disease (SGVHD) is a MHC class II-restricted T cell-mediated autoimmune syndrome that occurs following syngeneic bone marrow transplantation and the administration of cyclosporin (CsA). The present studies evaluated the V beta repertoire of T lymphocytes that mediate SGVHD. To facilitate analysis, SGVHD effector cells were adoptively transferred into thymectomized syngeneic recipients reconstituted with T cell-depleted bone marrow to provide an environment that allows for the selective clonal expansion of autoreactive T cells. Analysis of target tissues and PBL by reverse transcriptase PCR using oligonucleotide V beta-specific primers revealed a predominance of V beta 8.5+ T cells and a minor population expressing V beta 10. The majority of infiltrating lymphocytes in target tissues was confirmed to be V beta 8.5+ by in situ hybridization and by immunoperoxidase staining. A small population of V beta 10+ cells could also be detected. Furthermore, SGVHD effector T splenocytes depleted of lymphocytes expressing either the TCR-alpha beta or the V beta 8.5 determinant could not adoptively transfer SGVHD. Depletion of T cells expressing the V beta 10 determinant delayed the onset of this autoaggression syndrome. Subset analysis of the autoreactive T cell compartment revealed that the V beta 8.5 determinant was expressed on both CD4+ and CD8+ lymphocytes whereas the V beta 10 determinant was principally expressed on a minor population of CD4+ autoreactive T cells. These data were confirmed by limiting dilution analysis. Additional studies examining the effect of CsA on thymic differentiation revealed that although V beta 8.5 is not normally clonally deleted, there was a pronounced shift in the expression of this determinant between CD4 and CD8 single positive thymocytes, suggesting that CsA may inhibit normal positive selection processes for MHC class I and class II reactive T cells.

Myocarditis and cardiotropic viral infection associated with severe left ventricular dysfunction in late-stage infection with human immunodeficiency virus.

OBJECTIVES: The purpose of this study was to characterize the histologic and immunopathologic results of 37 endomyocardial biopsy samples from patients infected with human immunodeficiency virus type 1 (HIV-1) who were evaluated for unexplained global left ventricular dysfunction. BACKGROUND: Recent studies have identified a growing number of patients infected with HIV-1 who develop unexplained left ventricular dysfunction and congestive heart failure. Myocarditis has been confirmed at autopsy in small numbers of such patients, although a pathogenic opportunistic infectious agent can rarely be identified. METHODS: All patients had moderate to severe global left ventricular hypokinesia on two-dimensional echocardiography. Endomyocardial biopsy samples were evaluated by standard histologic studies, immunoperoxidase staining and in situ hybridization for cytomegalovirus and HIV-1 gene sequences. RESULTS: Twenty-eight patients presented with New York Heart Association functional class III or IV congestive heart failure. Four patients had myocarditis secondary to known etiologies (opportunistic infection n = 2; drug-induced hypersensitivity myocarditis n = 2). Of the remaining 33 samples, 17 (51%) showed histologic evidence of idiopathic active or borderline myocarditis. Immunohistologic findings revealed induced expression of major histocompatibility class I antigen on myocytes and increased numbers of infiltrating CD8+ T lymphocytes. Specific hybridization within myocytes was observed in 5 of 33 samples with the HIV-1 antisense riboprobe and in 16 of 33 samples with the cytomegalovirus immediate early (IE-2) antisense riboprobe. All but one patient with specific myocyte hybridization presented with congestive heart failure; all patients had myocarditis and CD4+ cell counts < 100/mm3. CONCLUSIONS: This study demonstrates that cardiotropic virus infection and myocarditis may be important in the pathogenesis of symptomatic HIV-associated cardiomyopathy.

Pulmonary hemorrhage as a cause of death in allogeneic bone marrow recipients with severe acute graft-versus-host disease.

Acute graft-versus-host disease (GVHD) has recently been associated with endothelial cell injury. The potential clinical significance was explored here in an autopsy review. Thirty-seven allogeneic bone marrow recipients were identified in the autopsy files at The Johns Hopkins Hospital with no evidence of systemic infection. Forty-one percent (15/37) of these patients were found to have extensive recent pulmonary hemorrhage at autopsy which was thought to have led to terminal respiratory failure and death. The 37 patients were divided into 2 groups: those with significant acute GVHD (stage 2 or greater) and those without GVHD (stage 0 or 1). Fifty-nine percent (10/17) of the patients with significant acute GVHD died of acute respiratory failure due to recent pulmonary hemorrhage as opposed to 25% (5/20) of those without acute GVHD (P = 0.032, Fisher's exact test). Terminal pulmonary hemorrhage was also associated with preparation for BMT, with 67% (12/18) of those prepared with total body irradiation (TBI) having pulmonary hemorrhage as opposed to 15% (3/19) of those prepared with chemotherapy using Busulphan (P = 0.002). There was no significant difference in posttransplant survival, engraftment, or final platelet count between the patients stratified by GVHD or preparative protocol. The data support a strong association between significant acute GVHD and terminal hemorrhage, as well a possible association between TBI and pulmonary hemorrhage. Analysis of variance demonstrates that GVHD and TBI are independently associated with increased pulmonary hemorrhage (P < 0.01 for GVHD, P < 0.001 for TBI). We propose that GVHD contributes to terminal pulmonary hemorrhage by injuring the endothelium. However, this association could also be a secondary effect, i.e., toxicity from therapy for GVHD, or an abnormality in cytokines or growth factors. The pathogenic relationship between significant GVHD and terminal hemorrhage is discussed briefly.

Myocarditis associated with doxorubicin cardiotoxicity.

In contrast to previous reports, the authors were impressed by the frequency of myocarditis in the endomyocardial biopsy specimens of patients treated with anthracyclines. To examine this, they reviewed the histologic and electron microscopic results and immunoperoxidase stains of myocardial biopsy specimens from 11 patients with doxorubicin cardiotoxicity grades 1.0-3.0. Immunoperoxidase stains for lymphocytes, macrophages, and endothelial cells and induced expression of Class II antigen were performed using the avidin-biotin complex procedure. A full panel of monoclonal antibodies was employed on fresh-frozen tissue; a smaller panel was used with formaldehyde-fixed paraffin-embedded material. Four of the 11 endomyocardial biopsy specimens showed myocarditis, and 2 showed borderline myocarditis by the Dallas criteria. The infiltrating lymphocytes were generally characterized as T lymphocytes and were associated with induced Class II antigen expression by arterial endothelial cells. In addition, foci of replacement fibrosis, suggesting a chronic process, were identified. Although this association does not prove a causal relationship, these results suggest that myocarditis can be a component of doxorubicin-induced myocardial injury.

Perforin expression localizing cytotoxic lymphocytes in the intimas of coronary arteries with transplant-related accelerated arteriosclerosis.

Accelerated arteriosclerosis is the major long-term complication of cardiac transplantation. It has been demonstrated recently that accelerated arteriosclerosis is caused, in part, by rejection-related, cell-mediated immunity. However, the role of cytotoxic T lymphocytes in this process is a subject of controversy. Perforin is a specific marker of functionally active cytotoxic lymphocytes because it is a functional component of the cytotoxic granules of these cells. We examined 11 coronary arteries from seven autopsied and four retransplanted heart transplant recipients for the presence of perforin-containing lymphocytes. Immunohistochemical stains for perforin were performed using a monoclonal antibody against human perforin. Eight of the 11 coronary arteries examined were found to contain perforin-positive cells. These perforin-positive cells were present in subendothelial spaces of the coronary arteries, and the staining seen was cytoplasmic and granular. The granules often were polarized to the endothelial surface. Furthermore, the cells identified were usually in close proximity to, or in direct contact with, coronary artery endothelial cells. These results suggest that cell-mediated endothelial injury by perforin-positive cytotoxic lymphocytes may contribute to the development of accelerated arteriosclerosis in heart transplant recipients.

In situ detection of human cytomegalovirus immediate-early gene transcripts within cardiac myocytes of patients with HIV-associated cardiomyopathy.

OBJECTIVE: Recent clinical and echocardiographic studies have identified dilated cardiomyopathy in 10-20% of HIV-infected adults. The purpose of this study was to determine the role of cardiotropic cytomegalovirus (CMV) infection in the development of HIV-associated cardiomyopathy. DESIGN: We generated sense and antisense digoxigenin-labeled riboprobes derived from the CMV immediate-early (IE) and delayed-early (DE) genes and applied them retrospectively to endomyocardial biopsy samples and control autopsy cardiac samples from HIV-infected patients. SETTING: Tertiary care, referral hospital. PATIENTS: Twelve consecutive HIV-infected patients with global left ventricular hypokinesis demonstrated on two-dimensional echocardiography; eight randomly selected control autopsy cardiac samples from HIV-infected patients without cardiac disease during life. MEASUREMENTS AND MAIN RESULTS: Of the 12 endomyocardial biopsy specimens, six (50%) were found to have specific myocyte nuclear and perinuclear hybridization for transcripts of the CMV IE gene, consistent with non-permissive or latent infection. Similar patterns were not found in any of the eight autopsy control samples. All six patients presented with unexplained congestive heart failure and had CD4 counts less than 100 x 10(6)/l; all six biopsy samples had immunohistochemical evidence of increased myocardial major histocompatibility complex (MHC) class I expression, a finding typical of non-HIV myocarditis. None of the endomyocardial biopsy samples had characteristic CMV inclusions and no specific hybridization was noted with the DE gene riboprobe, suggesting that no active viral DNA replication was present. Only two of the six patients with myocyte hybridization with the IE riboprobe had clinical evidence of solid organ infection with CMV at the time of cardiovascular presentation. CONCLUSIONS: This study is the first to demonstrate the expression of the IE gene of CMV within myocytes from HIV-infected patients with cardiomyopathy, suggesting a non-permissive infection of myocytes without classical intranuclear inclusions. Myocyte infection may be necessary to trigger cellular and humoral-mediated cardiac injury and may be best identified using in situ hybridization techniques.

Thalidomide for the treatment of chronic graft-versus-host disease.

BACKGROUND: Allogeneic bone marrow transplantation is an accepted therapy for hematologic cancer, aplastic anemia, and inherited immunodeficiencies. Chronic graft-versus-host disease (GVHD) is the principal complication in patients surviving more than 100 days. Thalidomide has been shown experimentally to be effective in treating GVHD. METHODS: We treated 23 patients with chronic GVHD refractory to conventional treatment and 21 patients with "high-risk" chronic GVHD (identified as having at least two of the following three risk factors: chronic GVHD that has evolved from acute GVHD, lichenoid skin or mucous-membrane changes, and hepatic dysfunction. Such patients have a high mortality rate.) with thalidomide in a dose that produced a plasma level of 5 micrograms per milliliter two hours after administration. Therapy was continued for three months after a complete response or for six months after a partial response. RESULTS: The overall actuarial survival of all enrolled patients was 64 percent. Survival was 76 percent among the patients receiving salvage therapy for refractory GVHD and 48 percent among those with high-risk GVHD. A complete response was observed in 14 patients, a partial response in 12 patients, and no response in 18. Side effects were minor, most notably sedation in almost all patients. CONCLUSIONS: In this preliminary trial, thalidomide appeared to be safe and effective for the treatment of chronic GVHD. A trial comparing thalidomide with prednisone in patients with newly diagnosed chronic GVHD will be required to demonstrate its relative efficacy.

t(11;18)(q21;q21) is a recurrent chromosome abnormality in small lymphocytic lymphoma.

We have identified two cases of previously untreated, small lymphocytic lymphoma with extranodal involvement, which had a reciprocal translocation, t(11;18)(q21;q21), as the sole cytogenetic abnormality. These two cases are remarkably similar to two previously reported cases carrying this translocation with regard to clinical features, cytogenetic abnormality, histologic subtype, and immunophenotype. Molecular genetic analysis of these two cases revealed clonal gene rearrangement of the IGH locus but only germline configuration of the BCL2 oncogene at 18q21 when probes and conditions that usually identify BCL2 rearrangement in lymphomas were used. Lymphomas bearing an (11;18) rearrangement appear to make up a phenotypically identifiable subgroup. Identification of the genes at the translocation breakpoints will be important.

Graft-specific MHC class II gene expression in response to allogeneic stimulus in heterotopic murine cardiac allografts.

Although, major histocompatibility complex (MHC) class II antigen expression in allografts is thoroughly studied, regulation of the genes for these antigens is not fully understood. The graft-specific MHC class II genes are potentially important in determining the immunogenicity of graft but their detection in a mixed-cell population such as in the allograft would require unambiguous differentiation of graft-specific class II expression from those in host lymphoid cells. With an oligonucleotide probe that specifically hybridizes to I-Ab mRNA from H-2k haplotype mice, we have studied I-A gene expression in cardiac allografts heterotopically transplanted from B10.Br (H-2k) to B10.D2 (H-2d) mice. Normal B10.Br hearts do not have appreciable I-Ab transcripts as determined with this probe, but 4 days after allografting, a substantial increase in I-Abk messenger RNA (mRNA) content was noted in the allografted hearts which persisted for the next 2 days and then decreased concomitant with destruction of the heart. The increase in I-Abk mRNA preceded the expression of surface Iak antigens on dendritic and endothelial cells in the allograft. These data indicate increased transcription of the I-Ab gene in cells of graft origin suggesting that transcriptional regulation is the initial mechanism for expression of class II genes in allografts. The sustained rise in graft-specific class II mRNA also seen in these allografts suggests that increased mRNA stability may be another mechanism for the increased density of class II antigens in allografts undergoing rejection.

Incidental myocarditis with intravenous drug abuse: the pathology, immunopathology, and potential implications for human immunodeficiency virus-associated myocarditis.

Human immunodeficiency virus (HIV)-associated myocarditis is well reported. Many of these patients are also intravenous (IV) drug abusers. This study examined sudden death victims from the Office of the Chief Medical Examiner, State of Maryland, for active myocarditis. A group of HIV-negative drug abusers is compared with a group of HIV-negative victims without any known risk factors for the acquired immunodeficiency syndrome. Using modified Dallas criteria combined with an avidin-biotin-labeled immunoperoxidase procedure, the cases were classified as active, borderline, or absent myocarditis. Serologic analysis for circulating antimyocardial antibody was performed along with phenotypic analysis of the infiltrate. Of the 15 IV drug abuse cases examined, none were positive for HIV (enzyme-linked immunosorbent assay and Western blot). With IV drug abuse as the primary risk factor, five cases demonstrated active myocarditis, while five others had borderline myocarditis. Only one of 10 low-risk patients had a lymphocytic infiltrate consistent with borderline myocarditis. Drug abuse-related myocarditis was associated with cardiac pathology resulting in sudden death in only one case. Antimyocardial antibodies were positive in four of the 15 IV drug abuse cases, including patients with active, borderline, and absent myocarditis. The phenotypic expression of the lymphocytic infiltrates was similar to the findings reported for idiopathic and HIV-related myocarditis (Am J Pathol 137:1365-1371, 1990). Toxicology studies did not implicate any particular drug of abuse, but the increased frequency of myocarditis observed may reflect complications of cocaine or the combined effects of opiates and cocaine. Intravenous drug abuse is an independent risk factor for myocarditis and must be taken into consideration in studies of HIV-associated myocarditis.

Enhancement of thymic recovery after cyclosporine by recombinant human growth hormone and insulin-like growth factor I.

Correlation of thymic changes with the development of CsA-associated syngeneic graft-versus-host disease (sGVHD) suggested that the development of tolerance depends on the prompt regeneration of the thymus after stopping CsA. Accordingly, we have tested recombinant human growth hormone (rhGH) and recombinant human insulin-like growth factor I (rhIGF-1) to determine if they accelerate reconstitution of the rat thymus after CsA-induced involution. After 14 days of CsA, the thymus has marked medullary involution but normally recovers fully in 6 weeks. In this study, LEW rats were injected with vehicle, rhGH, or rhIGF-1 for 21 days after stopping CsA and were examined. The vehicle-treated rats showed partial recovery with respect to Hassall's corpuscles, class II antigen expression, medullary size, medullary dendritic cells (DC), and T cell maturation. The mature thymocytes were predominantly CD8+ T cells. Both rhGH and rhIGF-1 induced significant thymic enlargement compared with the vehicle-treated rats. They also both significantly enhanced regeneration with respect to Hassall's corpuscles. The mature thymocyte population had significantly greater CD4+ cells. In addition, rhIGF-1 induced a significant improvement in the medullary size and medullary DC. While the medullae of a normal thymus are in intimate contact with cortical class II antigen, after CsA the cortex adjacent to the medulla is primarily class II antigen negative. RhGH significantly increased the class II antigen in the deep cortex while rhIGF-1 demonstrated a trend toward greater expression in this region (P = 0.06). We conclude that rhGH and rhIGF-1 accelerate thymic regeneration post-CsA. Further studies are now indicated to establish the potential for these factors to enhance the development of antigen-specific tolerance.

Prevention of syngeneic graft-versus-host disease by recovery of thymic microenvironment after cyclosporine.

Following a course of cyclosporine, syngeneic rat radiation chimeras consistently develop a GVHD-like syndrome. Correlation of the thymic immunopathology with conditions leading to syngeneic graft-versus-host disease (sGVHD) suggested the hypothesis that reconstitution of the normal thymic microenvironment after CsA is necessary for self-tolerance. When thymic regeneration is impaired, as in rats receiving previous mediastinal irradiation, then self-reactive effector cells are not regulated and proceed to damage the target tissues. Alternately, it could be argued that the observed thymic abnormalities are irrelevant to sGVHD. To test the primary hypothesis, post-CsA thymic reconstitution was prevented by total thymectomy in unirradiated rats. These rats consistently developed acute type sGVHD seen at 7 and 21 days post-CsA while rats from the CsA-treated sham thymectomy control group failed to develop sGVHD. Because thymectomy prior to CsA blocks sGVHD, most likely the peripheral effector cells in the post-CsA thymectomy group were derived from the CsA-altered thymus. The absence of sGVHD in the sham group indicates that the thymus led to active regulation of these cells after stopping CsA. If regeneration of the thymus restored only negative selection, then the sham thymectomy group should have also developed sGVHD. Flow cytometry and morphology of the spleen and lymph nodes demonstrated that the thymectomized rats, like CsA-treated radiation chimeras, experienced a significant delay in maturation of T cells following CsA. In contrast to the usual model in radiation chimeras, however, the post-CsA thymectomized rats did not convert to chronic type sGVHD. The importance of an abnormal thymus for this transition was confirmed in syngeneic radiation chimeras. Thymectomy after CsA in these rats also blocked the rapid transition to chronic sGVHD.

Prominent mononuclear cell infiltrate is characteristic of herpes esophagitis.

Diagnosis of herpes esophagitis is often difficult since the characteristic nuclear inclusions and/or multinucleate giant cells of herpes virus infection may be absent in endoscopic biopsy specimens. We have noted aggregates of large mononuclear cells with convoluted nuclei adjacent to infected epithelium in the exudates of herpetic esophagitis, and postulate that this is a characteristic inflammatory response to the virus. To test this hypothesis, we reviewed biopsies from 22 cases of ulcerative herpetic esophagitis and from 44 control cases of nonherpetic esophageal ulcers (including nine cases of candidal and five cases of bacterial esophagitis) that contained a quantifiable amount of exudate. The estimated percentage of mononuclear cells present in the specimens was ranked independently by two reviewers using coded photomicrographs of exudate. Wilcoxon's rank sum analysis demonstrated significant correlation between presence of herpes and increased mononuclear cells (P less than .0001). Only one of the 22 herpes cases did not show a prominent mononuclear cell infiltrate. Immunoperoxidase studies performed on Hollande-Bouin's-fixed paraffin-embedded material from 11 herpes cases showed strong staining of the mononuclear cells for KP-1 (CD68), indicating that the majority of these cells are macrophages. These findings suggest strongly that aggregates of macrophages are characteristic of the inflammatory response in ulcerative herpetic esophagitis. The presence of these mononuclear cells in a biopsy specimen that initially does not show herpetic inclusions warrants additional studies to rule out herpes virus infection.