Histological changes of bile duct in experimental graft-versus-host disease across minor histocompatibility barriers II. Electron microscopic observations

ABSTRACT— Electron microscopic features of intrahepatic bile ducts of experimental mouse graft-versus-host disease (GVHD) across minor histocompatibility barriers were studied for 14 months after transplantation. In GVHD mice, the bile duct epithelial layer was consistently infiltrated by lymphoid cells and often accompanied by polymorphonuclear leukocytes, monocytes and rarely by plasma cells. The epithelial cells in close contact with and in the vicinity of these infiltrated cells showed a variety of degenerative changes, including darkness of the cytoplasm and the nucleus with shrunken, irregular contours, increase in the amount of endoplasmic reticulum and number of mitochondria, and formation of intracytoplasmic vesicles and diverticula, cytoplasmic blebs, and apoptopic bodies. Lymphocytes were in close contact with epithelial cells through a number of point-contacts and located in the lateral intercellular spaces and/or between the basement membranes and the epithelial cells. The localization of infiltrating lymphocytes beneath the epithelial cells with conspicuous detachment from the basement membranes strongly suggested a link with subsequent epithelial cell injury and death. The lymphoid cells had irregular cytoplasmic projections which occasionally extended into spaces created by retractions of the epithelial cell membranes, reflecting an activation of the lymphocytes. These findings support the notion that the bile duct lesions in GVHD across minor histocompatibility barriers are mediated by specifically sensitized lymphocytes against epithelial cell membrane antigens. From our previous finding that a large majority of the infiltrating lymphocytes had a phenotype of helper/inducer T cells, a putative role of these lymphocytes in the induction of the bile duct lesions is discussed.     

Histological changes of the liver in experimental graft-versus-host disease across minor histocompatibility barriers. IV A study of lymphocyte-endothelial interaction

ABSTRACT— It has been reported in human hepatic graft-versus-host disease (GVHD) that an attachment of lymphocytes to vascular wall, the feature called “endothelialitis”, is the most important predictive histologic sign of GVHD. However, its precise nature and significance in GVHD are still unknown. We developed experimental mouse GVHD across minor histocompatibility barriers and examined the lesion during a 14-month period after transplantation. The lesion was transiently found, appearing first at 4 days after transplantation, reaching a maximal level at 2 weeks and disappearing 5 weeks after transplantation. Electron microscopically, an intimate interaction between lymphocyte and endothelial cell was demonstrated. Lymphocytes showed irregular cytoplasmic processes and pseudopods and were in close contact with endothelial cells. Lymphocytes frequently penetrated in between and under the endothehal cells, and migrated into the perivascular spaces. Immunohistochemical analysis revealed that the vast majority of lymphocytes attached to the endothelial cells are helper/inducer T cells, indicating the cardinal role of helper/inducer T cell in lymphocyte-endothelial cell interactions. These results, together with previous evidence of the presence of Ia antigens and an antigen-presenting ability of vascular endothelial cells, suggest that the attachment of lymphocytes to the vascular endothelial cells in the early course of GVHD may represent an in situ morphologic representation of antigen presentation by endothelial cells to helper T cells.     

Histological changes of the liver in experimental graft-versus-host disease across minor histocompatibility barriers. IV. A study of lymphocyte-endothelial interaction

It has been reported in human hepatic graft-versus-host disease (GVHD) that an attachment of lymphocytes to vascular wall, the feature called "endothelialitis", is the most important predictive histologic sign of GVHD. However, its precise nature and significance in GVHD are still unknown. We developed experimental mouse GVHD across minor histocompatibility barriers and examined the lesion during a 14-month period after transplantation. The lesion was transiently found, appearing first at 4 days after transplantation, reaching a maximal level at 2 weeks and disappearing 5 weeks after transplantation. Electron microscopically, an intimate interaction between lymphocyte and endothelial cell was demonstrated. Lymphocytes showed irregular cytoplasmic processes and pseudopods and were in close contact with endothelial cells. Lymphocytes frequently penetrated in between and under the endothelial cells, and migrated into the perivascular spaces. Immunohistochemical analysis revealed that the vast majority of lymphocytes attached to the endothelial cells are helper/inducer T cells, indicating the cardinal role of helper/inducer T cell in lymphocyte-endothelial cell interactions. These results, together with previous evidence of the presence of Ia antigens and an antigen-presenting ability of vascular endothelial cells, suggest that the attachment of lymphocytes to the vascular endothelial cells in the early course of GVHD may represent an in situ morphologic representation of antigen presentation by endothelial cells to helper T cells.     

Histological changes of the liver in experimental graft-versus-host disease across minor histocompatibility barriers. VII. A light and electron microscopic study of the large bile duct

ABSTRACT— Although morphologic changes of the intrahepatic bile ducts in graft-versus-host disease (GVHD) have been well studied, those of the large extrahepatic bile ducts in the porta hepatis or common bile ducts have not been so well elucidated. In the present study, pathologic changes of the extrahepatic bile ducts in experimental mouse GVHD across minor histocompatibility barriers were examined up to 14 months after transplantation. Mononuclear cell infiltration was most striking around 2 weeks after transplantation. Although it gradually decreased, infiltration persisted during the entire period of observation. Fibrous thickening and sclerosis of the bile duct wall continued over time following transplantation, especially 3 months after transplantation. The appearance was similar to sclerosing cholangitis, but obliteration of the lumen was not demonstrated. Electron microscopically, the bile duct epithelial layer was frequently infiltrated by lymphocytes, and often accompanied by polymorphonuclear leukocytes, monocytes, and rarely by plasma cells. The epithelial cells in close contact with and in the vicinity of these cells showed a variety of degenerative changes. These results suggest that not only the interlobular and/or small bile ducts but also the large hilar and extrahepatic bile ducts are involved in hepatic GVHD, and thus bile duct injury in GVHD may occur along the full length of the biliary tree.     

Histological changes of bile duct in experimental graft-versus-host disease across minor histocompatibility barriers. II. Electron microscopic observations

Electron microscopic features of intrahepatic bile ducts of experimental mouse graft-versus-host disease (GVHD) across minor histocompatibility barriers were studied for 14 months after transplantation. In GVHD mice, the bile duct epithelial layer was consistently infiltrated by lymphoid cells and often accompanied by polymorphonuclear leukocytes, monocytes and rarely by plasma cells. The epithelial cells in close contact with and in the vicinity of these infiltrated cells showed a variety of degenerative changes, including darkness of the cytoplasm and the nucleus with shrunken, irregular contours, increase in the amount of endoplasmic reticulum and number of mitochondria, and formation of intracytoplasmic vesicles and diverticula, cytoplasmic blebs, and apoptopic bodies. Lymphocytes were in close contact with epithelial cells through a number of point-contacts and located in the lateral intercellular spaces and/or between the basement membranes and the epithelial cells. The localization of infiltrating lymphocytes beneath the epithelial cells with conspicuous detachment from the basement membranes strongly suggested a link with subsequent epithelial cell injury and death. The lymphoid cells had irregular cytoplasmic projections which occasionally extended into spaces created by retractions of the epithelial cell membranes, reflecting an activation of the lymphocytes. These findings support the notion that the bile duct lesions in GVHD across minor histocompatibility barriers are mediated by specifically sensitized lymphocytes against epithelial cell membrane antigens. From our previous finding that a large majority of the infiltrating lymphocytes had a phenotype of helper/inducer T cells, a putative role of these lymphocytes in the induction of the bile duct lesions is discussed.     

Histological changes in the liver in experimental graft-versus-host disease across minor histocompatibility barriers. VI. A light and electron microscopic study of the periportal changes

Periportal changes of the liver in experimental graft-versus-host disease (GVHD) across minor histocompatibility barriers were investigated electron-microscopically for up to 14 months after bone marrow transplantation (BMT). In GVHD mice, periportal changes affecting the limiting plate of hepatocytes were relatively mild and, in general, classical piecemeal necrosis was rarely observed. However, around 2 weeks after transplantation disruption of the limiting plate of hepatocytes was transiently observed. At that time, lymphocytes invaded directly into the hepatic parenchyma and were in close contact with hepatocytes mainly through a number of point-contacts of cell membranes. Hepatocytes in close contact with lymphocytes showed minor degenerative changes under electron microscopy. On the other hand, periportal bile ductules and canals of Hering were constantly injured by inflammatory cells during the entire observation period up to 14 months after BMT. They were abutted by lymphocytes, together with other inflammatory cells including eosinophils, neutrophils, plasma cells and monocytes. Infiltration of inflammatory cells into the epithelial layer of the bile ductules and canals of Hering through the basement membrane was frequently found. Inflammatory cells were in contact with duct epithelial cells mainly through a number of point-contacts of cell membranes. Epithelial cells in contact with inflammatory cells exhibited a number of degenerative changes, including condensation of cytoplasm, irregular contour of nucleus, dilatation of endoplasmic reticulum, formation of cytoplasmic vesicles, focal cytoplasmic degeneration, and so on.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histological changes of the liver in experimental graft-versus-host disease across minor histocompatibility barriers. V. A light and electron microscopic study of the intralobular changes

Intralobular changes of the liver in experimental graft-versus-host disease (GVHD) across minor histocompatibility barriers were investigated for up to 14 months after bone marrow transplantation. Sinusoidal lymphocyte infiltration, and necrosis and degeneration of hepatocytes were evident by day 4 and reached a maximum level at 2 weeks after transplantation, then gradually decreased, but they persisted during the entire period of observation, indicating that more or less hepatocyte injury may persist continuously in hepatic GVHD. Piecemeal necrosis was transiently observed around 2 weeks after transplantation, in parallel with the peak of lymphocyte infiltration into the portal area. Similarly, central vein endothelialitis (attachment of lymphocytes to endothelial cells) was transiently observed with a peak activity at 2 weeks after transplantation. Mild centrilobular and portal fibroplasia were evident by 2 weeks after transplantation, but they hardly progressed and no cases developed liver cirrhosis. Frequently lymphocytes were found located beneath endothelial cells and attached to hepatocytes. Ultrastructural observation revealed that sinusoidal lymphocytes were occasionally in contact with endothelial cells by means of cytoplasmic pseudopods. Also lymphocytes were frequently in close contact with hepatocyte plasma membranes over short distances. Lymphocytes occasionally accompanied other inflammatory cells, such as eosinophilic leukocytes and mononuclear phagocytic cells. Hepatocytes in close contact with lymphocyte and other inflammatory cells showed a varying degree of degenerative changes, including condensation of cytoplasm and nucleus with irregular nuclear contours, dilatation of endoplasmic reticulum and mitochondria, formation of cytoplasmic vacuoles, and loss of microvilli.(ABSTRACT TRUNCATED AT 250 WORDS)     

Disparity for a newly identified minor histocompatibility antigen, HA-8, correlates with acute graft-versus-host disease after haematopoietic stem cell transplantation from an HLA-identical sibling

We recently identified a new minor histocompatibility antigen, termed HA-8, which is presented by human leucocyte antigen (HLA)-A*0201 or HLA-A*0202 and expressed ubiquitously among tissues. A retrospective analysis of 577 Caucasian patients with HLA-A*0201 or A*0202 who had received a haematopoietic stem cell transplant from a human leucocyte antigen (HLA)-identical sibling was conducted to determine whether HA-8 disparity correlated with clinical outcome. HA-8 disparity was detected in 72 recipients, and grades II-IV graft-versus-host disease (GVHD) occurred in 46 (64%), compared with 251 (50%) of the 503 patients without HA-8 disparity. After adjusting for known risk factors for acute GVHD, this difference was statistically significant (odds ratio, 1.8; 95% confidence interval, 1.0-3.1; P = 0.04). However, the hazards of clinical extensive chronic GVHD, overall mortality and recurrent malignancy were not statistically significantly different between the two groups. These data suggest that the increased risk of acute GVHD associated with recipient HA-8 disparity was not sufficient to change other clinical outcomes.     

Management of acute graft-versus-host disease

Acute graft-versus-host disease (GvHD) is a frequent complication of allogeneic haemopoietic stem cell transplantation (HSCT) and donor lymphocyte infusions (DLI). Its incidence and severity depends on several factors, such as prophylaxis method, donor/recipient matching, intensity of the conditioning regimen and composition of the graft. Significant progress has been made in recent years in understanding the pathogenesis of the disease, and some of these advances have been translated into clinical trials. First-line treatment of acute GvHD is based on corticosteroids, and produce sustained responses in 50-80% of patients depending on the initial severity. Non-responders are offered second-line therapy, with combinations of immunosuppressive agents, but 1-year survival is 30% in most large trials. New strategies explored include infusion of expanded mesenchymal stem cells (MSC), down regulation of antigen-presenting cells (APC) and suicide gene transduced T cells. Acute GvHD is complicated by severe immunodeficiency causing life-threatening infections. To date, GvHD has not been differentiated from the graft-versus-leukaemia effect. The present review will discuss some of these aspects.     

Cytokines are early diagnostic biomarkers of graft-versus-host disease in liver recipients

BACKGROUND: Graft-versus-host disease(GVHD) is associated with high mortality. Early diagnosis is essential to start treatment and to improve outcomes. Because of the inflammatory nature, we hypothesis that cytokine profile of patients with GVHD may serve as diagnostic markers. The present study was to evaluate the role of cytokine profile in the diagnosis of GVHD.METHODS: An immunoassay was used to detect 29 cytokines simultaneously in the serum; the measuring sensitivity of all cytokines was pg/m L. Healthy subjects undergoing annual routine physical examinations served as negative controls; 23 patients with hepatocellular carcinoma(HCC) who had undergone liver transplantation(the LT group) comprised the test subjects. A total of 22 kidney recipients with biopsyconfirmed GVHD(the RT group) were included for comparison. HCC patients with radical surgery(the HCC group, n=22) served as positive control. The liver contents of the three cytokines, IL-2, IL-18, and IFN-γ, were detected with immunohistochemistry. Serum granzyme B and perforin were measured by flow cytometry.RESULTS: Of the 29 cytokines, the levels of IL-2 and IL-18 were increased significantly in liver recipients with GVHD compared with healthy controls(P0.05). The serum levels of these three cytokines in the healthy, HCC, LT, and RT groups were IL-2: 0.90±0.02, 4.14±0.61, 5.10±0.89, and 1.48 ±0.09 pg/m L; IL-18: 80.61±9.35, 109.51±10.93, 230.11±12.92, and 61.98±7.88 pg/m L; IFN-γ: 24.06±3.88, 24.84±3.21, 40.37±5.88, and 15.33±4.72 pg/m L, respectively. Immunohistochemistry showed that these 3 cytokines expressions in the liver were parallel to the serum cytokine. After standard anti-GVHD treatment, the expressions of IL-2, IL-18, and IFN-γ were decreased in the liver(P0.05). Serum granzyme B and perforin were significantly increased in GVHD patients(P0.05). CONCLUSIONS: IL-2, IL-18 and IFN-γ were from liver and might serve as biomarkers for monitoring GVHD development and the effects of anti-GVHD treatment. Granzyme B and perforin may play a role in increasing IL-2, IL-18, and IFN-γ levels in GVHD patients.     

Role of extracorporeal photochemotherapy in patients with refractory chronic graft-versus-host disease

Recent studies suggest that extracorporeal photochemotherapy (ECP) may be beneficial in patients with steroid-refractory chronic graft-versus-host disease (cGvHD). However, it is not yet clear whether certain conditions, such as age, mode of onset of cGvHD etc., influence clinical response and whether certain affected organs are more sensitive to ECP than others. We analysed the main clinical and laboratory parameters related to evolution of the disease in 32 steroid-refractory cGvHD patients, to identify any useful response predictors to ECP. ECP affected the course of the disease positively in 78% (25/32) of our cases.     

Sirolimus in combination with tacrolimus and corticosteroids for the treatment of resistant chronic graft-versus-host disease

Chronic graft-versus-host disease (cGVHD) remains a major cause of morbidity and mortality in haematopoietic transplant recipients. Sirolimus is a macrocyclic triene antibiotic with immunosuppressive, antifungal and antitumour properties, that has activity in the prevention and treatment of acute GVHD. We conducted a phase II trial of sirolimus combined with tacrolimus and methylprednisolone in patients with steroid-resistant cGVHD. Thirty-five patients who developed GVHD after day 100 post-transplant were studied. Six patients had a complete response and 16 a partial response with an overall response rate of 63%. Major adverse events related to the combination of tacrolimus and sirolimus were hyperlipidaemia, renal dysfunction and cytopenias. Four patients had thrombotic microangiopathy (TMA) and 27 (77%) had infectious complications. The median survival for the whole group was 15 months. A significantly better outcome was observed in patients with a platelet count > or = 100 x 10(9)/l, as well as in those with true chronic manifestations of GVHD compared to those with acute GVHD beyond day 100. Controlled trials comparing this approach with alternative strategies to determine which can best achieve the goal of GVHD-free survival are warranted.     

Disparity for the minor histocompatibility antigen HA-1 is associated with an increased risk of acute graft-versus-host disease (GvHD) but it does not affect chronic GvHD incidence, disease-free survival or overall survival after allogeneic human leucocyte antigen-identical sibling donor transplantation

Disparity for the minor histocompatibility antigen HA-1 between patient and donor has been associated with an increased risk of acute graft-versus-host disease (GvHD) after allogeneic human leucocyte antigen (HLA)-identical sibling donor stem cell transplantation (SCT). However, no data concerning the impact of such disparity on chronic GvHD, relapse or overall survival are available. A retrospective multicentre study was performed on 215 HLA-A2-positive patients who received an HLA-identical sibling SCT, in order to determine the differences in acute and chronic GvHD incidence on the basis of the presence or absence of the HA-1 antigen mismatch. Disease-free survival and overall survival were also analysed. We detected 34 patient-donor pairs mismatched for HA-1 antigen (15.8%). Grades II-IV acute GvHD occurred in 51.6% of the HA-1-mismatched pairs compared with 37.1% of the non-mismatched. The multivariate logistic regression model showed statistical significance (P: 0.035, OR: 2.96, 95% CI: 1.07-8.14). No differences were observed between the two groups for grades III-IV acute GvHD, chronic GvHD, disease-free survival or overall survival. These results confirmed the association between HA-1 mismatch and risk of mild acute GvHD, but HA-1 mismatch was not associated with an increased incidence of chronic GvHD and did not affect relapse or overall survival.     

Prevention of the acute graft-versus-host disease (GVHD) in rats by the immunomodulating drug leflunomide

Summary The grafting of immunocompetent allogeneic cells into MHC-discordant, genetically nonresponsive F₁ hybrids of inbred rat strains consistently leads to an acute, lethal graft-versus-host disease (GVHD). The novel immunomodulating drug leflunomide, which has been shown to be efficacious in animal models of autoimmunity and adverse transplantation reactions, was studied in a rat model of GVHD. It was found that this drug not only was a powerful agent to prevent this otherwise terminal disorder, but was also proficient when used as a therapy of an established GVHD. Since leflunomide has been shown to be efficacious and safe in patients with chronic rheumatoid arthritis, it would also be reasonable to investigate this drug in clinical trials for bone marrow transplantation and GVHD in human beings.This work was supported by the Wilhelm Sander-Stiftung Neuburg a. d. Donau, Number 90.059.1     

Factors for graft-versus-host disease after donor lymphocyte infusions with an escalating dose regimen: lack of association with cell dose

We investigated the risk factors for graft-versus-host disease (GVHD) in 82 patients treated with donor lymphocyte infusions (DLI) using an escalating dose regimen for chronic myeloid leukaemia in relapse following conventional allografting. Two factors emerged as predictors of both acute and chronic GVHD: the infusion of male recipients with lymphocytes from a female donor and the interval between transplant and last DLI, but only the first remained significant at multivariate analysis. Surprisingly, lymphocyte dose did not influence the incidence of GVHD. Our results suggest that DLI can be given in large cell doses without increasing the risk of GVHD.     

CD4+ T cells appear capable of initiating graft-versus-host disease across non-major histocompatibility complex (MHC) barriers in man

Twelve patients with haematological malignancy received cyclophosphamide 120 mg/kg, fractionated total body irradiation 12 Gy, oral cyclosporin and an HLA-identical sibling marrow transplant depleted of T cells by incubation with monoclonal antibodies directed against the CD2 and CD8 antigens and rabbit complement. The phenotype of the residual T cells in the donor marrow inocula was CD3+, CD4+, CD8-. To exclude the possibility that this represented modulation or blocking of the CD8 antigen, T-depleted and non-depleted marrow aliquots from these donors were bulk-cultured for 10 days with phytohaemagglutinin and interleukin-2. Even after this attempted expansion, only a small proportion of cultured T cells from the depleted aliquots (in contrast to the non-depleted aliquots) expressed the CD8 antigen. Since all patients receiving CD3+, CD4+, CD8- marrow developed mild or moderate acute graft-versus-host disease (GVHD), we conclude that CD4+ T cells are capable of initiating acute GVHD across non-MHC barriers in man.     

Extracorporeal photopheresis induces apoptosis in the lymphocytes of cutaneous T-cell lymphoma and graft-versus-host disease patients

Extracorporeal photopheresis (ECP) is used in the treatment of T-cell-mediated disorders. However, the mechanism by which ECP achieves its effect remains illusive. Over recent years the ability of ECP to induce apoptosis has been demonstrated by cell culture experiments and retrospective histological analysis. We investigated if apoptosis could be determined in samples tested ex vivo from the UVAR:ECP system. Lymphocytes from 11 patients (six with cutaneous T-cell lymphoma, four with graft-versus-host disease, and one with scleredema) were isolated at three stages of the ECP process: immediately before ECP treatment, from the first buffy coat collected, and post UV irradiation, prior to re-infusion. Using flow cytometry each stage was tested for the early apoptotic markers; Annexin V, ApoptestTM and Carboxy-SNARF-1-AM. Comparisons of the pre-ECP and pre-infusion samples demonstrated a significant increase in apoptotic lymphocytes for all three flow cytometric techniques (P < 0.01). Increases between the pre-ECP and first buffy coat, used as a measure of the extracorporeal manipulation, were much lower. These results demonstrate that ECP directly induces significant levels of apoptosis in lymphocytes of CTCL, GvHD and scleredema patients. The apoptosis of these lymphocytes may contribute to the ECP effect.     

Rectal biopsy,rather than ileal,is appropriate to confirm the diagnosis of early gastrointestinal graft-versus-host disease

Abstract

Objective. Once gastrointestinal (GI) graft-versus-host disease (GVHD) occurs after hematopoietic stem cell transplantation, it may be life-threatening. Therefore, an earlier accurate diagnosis of macroscopic and microscopic features using an appropriate modality improves the prognosis of patients with suspected GI-GVHD. Patients and methods. In patients experiencing watery diarrhea within 100 days after hematopoietic stem cell transplantation, we evaluated the severity of mucosal injury at the proximal ileum, terminal ileum, and rectum according to previously reported criteria using transanal single balloon endoscopy. GI-GVHD was diagnosed by the presence of gland apoptosis without inflammatory or infectious factors in the biopsied specimens obtained from their respective site regardless of the mucosal lesion. Results. Consecutive suspected GI-GVHD patients with watery diarrhea (11 men and 5 women, mean age: 45.6 years, coexistent symptoms: nausea [38%] and exanthema [69%]) were enrolled. GI-GVHD was identified pathologically in 11 patients (69%), all of whom had pathological findings of GI-GVHD at the rectum. However, eight patients (73%) had pathological findings of GI-GVHD at both the ileum and the rectum and none had pathological findings of GI-GVHD at the ileum alone. The accuracies for a pathological diagnosis of GI-GVHD based on endoscopic features were 44%, 44%, and 38% at the proximal ileum, terminal ileum, and rectum, respectively. The severity of mucosal injury had no association with the diagnostic rate of pathological GI-GVHD at any site. Conclusions. A pathological evaluation of the rectum but not the ileum may be important and useful for the accurate diagnosis of early GI-GVHD.     

An alpha-defensin gene single nucleotide polymorphism modulates the gut microbiota and may alter the risk of acute graft-versus-host disease

We previously reported a protective association between single nucleotide polymorphisms (SNPs; rs4415345G and rs4610776A alleles) of Paneth cell α-defensin-5 against acute graft-versus-host disease (aGVHD). Because dysbiosis has been associated with aGVHD, we hypothesized that these SNPs may have a gut microbiota signature. In Lasso regression analysis of 248 healthy individuals, rs4415345G was associated with a higher abundance of Odoribacter splanchnicus, an anaerobic butyrogenic commensal. In multivariable analysis of data from 613 allogeneic haematopoietic cell transplant recipients, peri-engraftment presence of O. splanchnicus was associated with ~50% lower risk for grade II–IV aGVHD (hazard ratio 0·53, 95% confidence interval 0·28–1·00, P = 0·05). O. splanchnicus may protect rs4415345G individuals against aGVHD.