Machine learning and single cell RNA sequencing analysis identifies regeneration-related hepatocytes and highlights a Birc5-related model for identifying cell proliferative ability.

BACKGROUND: Partial hepatectomy (PHx) has been shown to induce rapid regeneration of adult liver under emergency conditions. Therefore, an in-depth investigation of the underlying mechanisms that govern liver regeneration following PHx is crucial for a comprehensive understanding of this process. METHOD: We analyzed scRNA-seq data from liver samples of normal and PHx-48-hour mice. Seven machine learning algorithms were utilized to screen and validate a gene signature that accurately identifies and predicts this population. Co-immunostaining of zonal markers with BIRC5 to investigate regional characteristics of hepatocytes post-PHx. RESULTS: Single cell sequencing results revealed a population of regeneration-related hepatocytes. Transcription factor analysis emphasized the importance of Hmgb1 transcription factor in liver regeneration. HdWGCNA and machine learning algorithm screened and obtained the key signature characterizing this population, including a total of 17 genes and the function enrichment analysis indicated their high correlation with cell cycle pathway. It is note-worthy that we inferred that Hmgb1 might be vital in the regeneration-related hepatocytes of PHx_48h group. Parallelly, Birc5 might be closely related to the regulation of liver regeneration, and positively correlated with Hmgb1. CONCLUSIONS: Our study has identified a distinct population of hepatocytes that are closely associated with liver regeneration. Through machine learning algorithms, we have identified a set of 17 genes that are highly indicative of the regenerative capacity of hepatocytes. This gene signature has enabled us to assess the proliferation ability of in vitro cultured hepatocytes using sequencing data alone.

A Five-LLPS Gene Risk Score Prognostic Signature Predicts Survival in Hepatocellular Carcinoma.

Background: Primary liver cancer, dominated by hepatocellular carcinoma (HCC), is one of the most common cancer types and the third leading cause of cancer death in 2020. Previous studies have shown that liquid-liquid phase separation (LLPS) plays an important role in the occurrence and development of cancer including HCC, but its influence on the patient prognosis is still unknown. It is necessary to explore the effect of LLPS genes on prognosis to accurately forecast the prognosis of HCC patients and identify relevant targeted therapeutic sites. Methods: Using The Cancer Genome Atlas dataset and PhaSepDB dataset, we identified LLPS genes linked to the overall survival (OS) of HCC patients. We applied Least Absolute Shrinkage and Selection Operator (LASSO) Cox penalized regression analysis to choose the best genes for the risk score prognostic signature. We then analysed the validation dataset and evaluated the effectiveness of the risk score prognostic signature. Finally, we performed quantitative real-time PCR experiments to validate the genes in the prognostic signature. Results: We identified 43 differentially expressed LLPS genes that were associated with the OS of HCC patients. Five of these genes (BMX, FYN, KPNA2, PFKFB4, and SPP1) were selected to generate a prognostic risk score signature. Patients in the low-risk group were associated with better OS than those in the high-risk group in both the training dataset and the validation dataset. We found that BMX and FYN had lower expression levels in HCC tumour tissues, whereas KPNA2, PFKFB4, and SPP1 had higher expression levels in HCC tumour tissues. The validation demonstrated that the five-LLPS gene risk score signature has the capability of predicting the OS of HCC patients. Conclusion: Our study constructed a five-LLPS gene risk score signature that can be applied as an effective and convenient prognostic tool. These five genes might serve as potential targets for therapy and the treatment of HCC.

Splenic Artery Steal Syndrome in Patients with Liver Cirrhosis: A Retrospective Clinical Study.

BACKGROUND Splenic artery steal syndrome (SASS) can aggravate liver damage in patients with cirrhosis. This study explored whether SASS could be an effective therapeutic target for improving hepatic artery perfusion and liver function in patients with decompensated cirrhosis. MATERIAL AND METHODS Based on inclusion and exclusion criteria, 87 patients with hepatitis B cirrhosis and portal hypertension hypersplenism admitted to our General Surgery Department for splenectomy and pericardial devascularization surgery were selected. A total of 35 cases met the diagnostic criteria of SASS and were assigned to the SASS group; the remaining 52 cases were assigned to the control group. The indicators before, during, and after surgery were compared between the 2 groups. RESULTS There were no significant differences in preoperative and intraoperative indicators between SASS group and control group (P>0.05). The MELD score 7 days after surgery and the hepatic artery diameter and hepatic artery velocity 14 days after surgery in both groups were significantly better than before surgery. The MELD score 7 days after surgery in the SASS group was significantly better than that in the control group, and the hepatic artery diameter and hepatic artery velocity 14 days after surgery in the SASS group were significantly better than those in the control group (P<0.05). CONCLUSIONS Splenectomy and pericardial devascularization surgery was an effective treatment to redirect blood flow to the hepatic artery for cirrhotic patients diagnosed with SASS. The introduction of cirrhotic SASS into clinical practice may benefit more patients with cirrhotic portal hypertension and hypersplenism.

A Multiparametric Nomogram for Predicting Delayed Graft Function in Adult Recipients of Pediatric Donor Kidneys.

BACKGROUND: Delayed graft function (DGF) is one of the most common postoperative complications after kidney transplantation. The ability to predict DGF after transplantation can greatly aid clinical decision-making. Several models have been proposed to predict DGF in adult recipients of adult donor kidneys, but there is currently no model to predict DGF in adult recipients of pediatric donor transplants. Therefore, based on our medical records, we retrospectively investigated the pretransplant risk factors of DGF in transplants from pediatric donors to adult recipients. METHODS: Our center is in compliance with national laws, the Declaration of Istanbul, and the Helsinki Congress. The donors used by us were organs donated after the death of citizens and the participants were neither paid nor coerced. A retrospective review of 84 adult patients who received pediatric donor kidneys at a single center from April 4, 2015 to November 17, 2021 was conducted to investigate the pretransplant risk factors for the development of DGF. RESULTS: DGF was observed in 45 of 68 patients (66.17%) in the training group and 9 of 16 patients (56.25%) in the validation group. Multivariate logistic analysis showed that kidney donor profile index, cold ischemia time, number of human leukocyte antigen mismatches, and pretransplant dialysis duration were significant independent risk factors for DGF. By integrating these 4 factors, we constructed a nomogram model to predict DGF. According to the prediction model, the area under the curve of DGF of the training group and validation group was 0.899 and 0.905, respectively. CONCLUSION: We have constructed a novel, reliable, and accurate visual nomogram that provides a practical tool for predicting DGF in adult recipients of pediatric donor kidneys.

A gene-based risk score model for predicting recurrence-free survival in patients with hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) remains the most frequent liver cancer, accounting for approximately 90% of primary liver cancers worldwide. The recurrence-free survival (RFS) of HCC patients is a critical factor in devising a personal treatment plan. Thus, it is necessary to accurately forecast the prognosis of HCC patients in clinical practice. METHODS: Using The Cancer Genome Atlas (TCGA) dataset, we identified genes associated with RFS. A robust likelihood-based survival modeling approach was used to select the best genes for the prognostic model. Then, the GSE76427 dataset was used to evaluate the prognostic model's effectiveness. RESULTS: We identified 1331 differentially expressed genes associated with RFS. Seven of these genes were selected to generate the prognostic model. The validation in both the TCGA cohort and GEO cohort demonstrated that the 7-gene prognostic model can predict the RFS of HCC patients. Meanwhile, the results of the multivariate Cox regression analysis showed that the 7-gene risk score model could function as an independent prognostic factor. In addition, according to the time-dependent ROC curve, the 7-gene risk score model performed better in predicting the RFS of the training set and the external validation dataset than the classical TNM staging and BCLC. Furthermore, these seven genes were found to be related to the occurrence and development of liver cancer by exploring three other databases. CONCLUSION: Our study identified a seven-gene signature for HCC RFS prediction that can be used as a novel and convenient prognostic tool. These seven genes might be potential target genes for metabolic therapy and the treatment of HCC.

FYN is required for ARHGEF16 to promote proliferation and migration in colon cancer cells.

ARHGEF16 is a recently identified Rho-family guanine nucleotide exchange factor (GEF) that has been implicated in the activation of Rho-family GTPases such as Rho G, Rac, and Cdc42. However, its functions in colon cancer cell proliferation and migration are not well understood. In this study, we showed that ARHGEF16 was highly expressed in clinical specimens of colon cancer. In colon cancer cells, ARHGEF16-stimulated proliferation and migration in vitro and in vivo. Furthermore, we identified a nonreceptor tyrosine kinase, FYN, as a novel partner of ARHGEF16. Knocking down FYN expression decreased ARHGEF16 protein level in colon cancer cells. We further demonstrated that ARHGEF16-induced colon cancer cell proliferation and migration were dependent on FYN since knockdown FYN abolished the ARHGEF16-induced proliferation and migration of colon cancer cells. The FYN-ARHGEF16 axis mediates colon cancer progression and is a potential therapeutic target for colon cancer treatment.

The critical role of dysregulated Hh-FOXM1-TPX2 signaling in human hepatocellular carcinoma cell proliferation.

BACKGROUND: Aberrant activation of the Hedgehog (Hh) signaling pathway is frequently observed in hepatocellular carcinoma (HCC), nevertheless, the precise molecular mechanism remains unclear. Forkhead box M1 (FOXM1), a target of the Hh pathway, is a key oncofetal transcription factor and a master cell cycle regulator. Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is an oncogene critical for mitosis. However, how these molecular events affect HCC progression remains unclear. METHODS: Realtime PCR, immunohistochemistry, western blotting, and analyses of datasets TCGA and Gene Expression Omnibus (GEO) were conducted to assess the expression of TPX2 and FOXM1 at the mRNA and protein levels in HCC samples or HCC cells. Expression and knockdown of TPX2 and FOXM1 were performed to assess their role in regulating HCC cell proliferation in vitro and in vivo. Dual luciferase report assay and chromosome immunoprecipitation (ChIP) were investigated to seek the FOXM1 binding sites in the promoter of TPX2. RESULTS: Specific antagonists (cyclopamine and GANT61) of the Hh pathway down-regulated TPX2, whereas activation of Hh signaling stimulated TPX2 expression. Furthermore, TPX2 over-expression accelerated HCC cell proliferation when upstream events of Hh signaling were inhibited, and TPX2 knockdown significantly alleviated Sonic Hh ligand (Shh)-induced HCC cell proliferation. Reporter assays and ChIP showed that FOXM1 bound to the TPX2 promoter, confirming that TPX2 is a direct downstream target of FOXM1. Xenograft model further verified the cell function and expression regulation of TPX2 and FOXM1 in vivo. Furthermore, FOXM1 regulated TPX2 activity to drive HCC proliferation. Immunohistochemical (IHC) analysis indicated that FOXM1 and TPX2 were highly-expressed in HCC samples and cohort study revealed that FOXM1 and TPX2 may act as negative predictors for the prognosis of patients with HCC. CONCLUSIONS: TPX2 acts as a novel downstream target and effector of the Hh pathway, and Hh signaling contributes to HCC proliferation via regulating the FOXM1-TPX2 cascade, suggesting that this signaling axis may be a novel therapeutic target for HCC.

Identification of potential key genes and pathways in hepatitis B virus-associated hepatocellular carcinoma by bioinformatics analyses.

Chronic hepatitis B virus (HBV) is one of the leading causes of hepatocellular carcinoma (HCC). The precise molecular mechanisms by which HBV contributes to HCC development are not fully understood. The key genes and pathways involved in the transformation of nontumor hepatic tissues into HCC tissues in patients with HBV infection are essential to guide the treatment of HBV-associated HCC. Five datasets were collected from the Gene Expression Omnibus database to form a large cohort. Differentially expressed genes (DEGs) were identified between HCC tissues and nontumor hepatic tissues from HBV-infected patients using the 'limma' package. The top 50 upregulated and top 50 downregulated DEGs in HCC vs. nontumor tissues were demonstrated in subsets by heat maps. Based on the DEGs, Gene Ontology functional and Kyoto Encyclopedia of Genes and Genomes pathways enrichment analyses were performed. Several key pathways of the up- and downregulated DEGs were identified and presented by protein-protein interaction (PPI) networks. A total of 1,934 DEGs were identified. The upregulated DEGs were primarily associated with the 'cell cycle'. Among the DEGs enriched in the 'cell cycle' pathway, 6 genes had a log2-fold change >2: SFN, BUB1B, TTK, CCNB1, CDK1 and CDC20. The downregulated DEGs were primarily associated with the metabolic pathways, such as 'carbon metabolism', 'glycine, serine and threonine metabolism', 'tryptophan metabolism', 'retinol metabolism' and 'alanine, aspartate and glutamate metabolism'. The DEGs in the 'cell cycle' and 'metabolic pathways' were presented by the PPI networks respectively. Overall, the present study provides new insights into the specific etiology of HCC and molecular mechanisms for the transformation of nontumor hepatic tissues into HCC tissues in patients with a history of HBV infection and several potential therapeutic targets for targeted therapy in these patients.

FOXM1 promotes hepatocellular carcinoma progression by regulating KIF4A expression.

BACKGROUND: Forkhead box M1 (FOXM1) is a proliferation-associated transcription factor of the forkhead box proteins superfamily, which includes four isoforms FOXM1a, b, c, and d. FOXM1 has been implicated in hepatocellular carcinoma (HCC) progression, but the underlying molecular mechanism remains elusive. In this study, we aim to clarify the molecular basis for FOXM1-mediated HCC progression. METHODS: Bioinformatic analysis was used to explore the differentially expressed genes predicting HCC proliferation. The expression of FOXM1 and kinesin family member (KIF)4A was confirmed by western blotting and immunohistochemistry in HCC tissues. Kaplan-Meier survival analysis was conducted to analyze the clinical impact of FOXM1 and KIF4A on HCC. The effect of FOXM1 on the regulation of KIF4A expression was studied in cell biology experiments. The interaction between KIF4A and FOXM1 was analyzed by chromatin immunoprecipitation and luciferase experiments. A series of experiments was performed to explore the functions of FOXM1/KIF4A in HCC progression, such as cell proliferation, cell growth, cell viability, and cell cycle. A xenograft mouse model was used to explore the regulatory effect of FOXM1-KIF4A axis on HCC tumor growth. RESULTS: FOXM1 and KIF4A were overexpressed in human primary HCC tissues compared to that in matched adjacent normal liver tissue and are significant risk factors for HCC recurrence and shorter survival. We found that KIF4A was dominantly regulated by FOXM1c among the four isoforms, and further identified KIF4A as a direct downstream target of FOXM1c. Inhibiting FOXM1 decreased KIF4A expression in HCC cells, whereas its overexpression had the opposite effect. FOXM1-induced HCC cell proliferation was dependent on elevated KIF4A expression as KIF4A knockdown abolished FOXM1-induced proliferation of HCC cells both in vitro and in vivo. CONCLUSION: The FOXM1-KIF4A axis mediates human HCC progression and is a potential therapeutic target for HCC treatment.

Tumor repressor gene chondroadherin oppose migration and proliferation in hepatocellular carcinoma and predicts a good survival.

The molecular that used as prognosis and potential therapy target is urgently needed in hepatocellular carcinoma (HCC). In current work, we found the expression of CHAD (chondroadherin) was significantly reduced in hepatocellular carcinoma compared to the normal tissue, on both mRNA and protein levels, in three independent datasets. Survival analysis was implemented on these datasets, and low expression of CHAD was found to be significantly associated with poor survival. Furthermore, metastasis-averse HCC and metastasis-incline HCC group comparison, and protein abundance evaluation of normal-tumor-portal vein tumor thrombus pairs indicate that metastatic tendentiousness is reduced along with CHAD abundance. Correlation analysis was also carried out and CHAD was shown to be significantly associated with differentiation and metastasis. Multivariable cox regression analysis showed that CHAD expression is more important for prognosis, compared to the other clinical indicators. To facilitate the utilization of CHAD clinically, a nomogram was plotted to estimate the three-year survival rate. Functional assays testing the migration and proliferation ability following knock down of CHAD in two cell lines, SMMC7721 and HCCLM3, were performed and discovered that reduction of CHAD level significantly enhance both proliferation and migration in both cell lines. Gene Set Enrichment Analysis (GSEA) comparing the CHAD-low and CHAD-high group showed that KEGG signaling pathways including "focal adhesion", "ECM receptor interaction", and "regulation of actin cytoskeleton" were significantly enriched. In conclusion, as a potential prognostic biomarker, tumor suppressor gene CHAD represses migration and proliferation of hepatocellular carcinoma cells, probability via mediating cell-cell adhesion.

Aberrantly activated Gli2-KIF20A axis is crucial for growth of hepatocellular carcinoma and predicts poor prognosis.

UNLABELLED: Glioma-associated oncogene 2 (Gli2), a primary transcriptional regulator of Hedgehog (Hh) signaling, is essential for hepatocellular carcinoma (HCC) growth and survival. However, the underlying molecular mechanism and crucial downstream targets of Gli2 in human HCC are not fully understood. Here, we report the identification of kinesin family member 20A (KIF20A) as a novel downstream target of Gli2, which is important for HCC proliferation and tumor growth. Inhibition of Hh signaling leads to a remarkable decrease of KIF20A expression in HCC cells, whereas overexpression of Gli2 elevates KIF20A expression by activating Forkhead Box M1 (FoxM1)-MMB complex-mediated transcription of this kinesin gene. Gli2-induced HCC cell growth requires enhanced expression of KIF20A, and knockdown of Gli2 or KIF20A represses the proliferation of HCC cells in vitro and in vivo. Correlated with these results, analyses of clinical HCC samples show that Gli2, FoxM1 and KIF20A are highly elevated in primary HCC samples and represent significant risk factors for HCC recurrence and survival. CONCLUSION: KIF20A is an important downstream target gene of Hh signaling. And, the Gli2-KIF20A axis is essential for the proliferation and growth of human HCC cells. Our study also suggests Gli2-KIF20A axis as a potential target for future therapeutic intervention and as an independent prognostic biomarker for HCC.

Giant biliary cystadenoma complicated with polycystic liver: a case report.

Biliary cystadenoma (BCA) is a rare hepatic neoplasm. Although considered a benign cystic tumor of the liver, BCA has a high risk of recurrence with incomplete excision and a potential risk for malignant degeneration. Correct diagnosis and complete tumor excision with negative margins are the mainstay of treatment. Unfortunately, due to the lack of presenting symptoms, and normal laboratory results in most patients, BCA is hard to distinguish from other cystic lesions of the liver such as biliary cystadenocarcinoma, hepatic cyst, hydatid cyst, Caroli disease, undifferentiated sarcoma, intraductal papillary mucinous tumor, and hepatocellular carcinoma. Ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI) may be necessary. They demonstrate intrahepatic cystic lesions with features such as mural nodules, varying wall thickness, papillary projections, and internal septations. Nevertheless, surgery is still the only means of accurate diagnosis. Definitive diagnosis requires histological examination following formal resection. We describe a 57-year-old woman initially diagnosed with polycystic liver who was subsequently diagnosed with giant intrahepatic BCA in the left hepatic lobe. This indicates that both US physicians and hepatobiliary specialists should attach importance to hepatic cysts, and CT or MRI should be performed for further examination when a diagnosis of BCA is suspected.

Late CD8+ T cell-dependent xenoantibody production in innate tolerant nude rats after hamster islet grafting but not after hamster heart grafting.

BACKGROUND: Xenograft rejection can be provoked by both the innate and adaptive immune compartments and close reciprocal interactions exist between these two systems. We investigated the interdependent roles of T and B lymphocytes in vascularized (heart) and cellular (islet) xenograft rejection in a model with established xeno-nonreactivity of the innate immune system. METHODS: Specific innate xenotolerance was induced in nude rats bearing either a hamster heart or a hamster pancreatic islet graft by a tolerizing regimen consisting of donor antigen infusion, temporary natural killer cell depletion and a 4-week administration of leflunomide. One month after transplantation, syngeneic CD4 and CD8 T cells were adoptively transferred. RESULTS: Both vascular and cellular xenografts were rejected after CD4 T cell reconstitution, corresponding with production of high IgM and IgG xenoantibody titers. Deposition of xenoantibodies and complement was seen in the heart but not in the islet xenografts. After infusion of CD8 T cells, xenohearts underwent a delayed type of rejection without xenoantibody production and xenoislets were not rejected. In xenoislet recipients, CD8 dependent B cells were not tolerized, resulting in the production of IgG xenoantibodies belonging to Th2-dependent isotypes, known not to cause graft rejection, and deposited at the graft implantation site. CONCLUSIONS: We conclude that distinct mechanisms of immune activation underlie xenogeneic reactions against vascular and cellular grafts.

Occurrence of autoimmunity after xenothymus transplantation in T-cell-deficient mice depends on the thymus transplant technique.

Xenothymus transplantation under the kidney capsule in athymic rodents frequently leads to multiorgan autoimmunity. Herein, we explore whether this is an intrinsic risk of xenothymus grafting or whether it depends on the transplant technique. We developed a new technique of "venous pouch" thymus grafting (heart-xenothymus) and compared this with the conventional kidney subcapsular technique (kidney-xenothymus) in a rat-into-nude-mouse model. Whereas lethal autoimmunity developed in 90% of kidney-xenothymus recipients, all heart-xenothymus grafted mice remained completely healthy. Autoimmunity in heart-xenothymus recipients was absent despite a significantly improved T-cell generation and was associated with significantly higher CD4+CD25+ T-cell frequencies and CD4+CD25+ cell Foxp3 mRNA levels than those observed in kidney-xenothymus recipients. In conclusion, we describe a novel vascular pouch technique of xenothymus transplantation that prevents the development of autoimmunity in nude mice. Our data further suggest that prevention of autoimmunity is related to a superior development of regulatory T-cells.

Rapidly induced, T-cell independent xenoantibody production is mediated by marginal zone B cells and requires help from NK cells.

Xenoantibody production directed at a wide variety of T lymphocyte-dependent and T lymphocyte-independent xenoantigens remains the major immunologic obstacle for successful xenotransplantation. The B lymphocyte subpopulations and their helper factors, involved in T-cell-independent xenoantibody production are only partially understood, and their identification will contribute to the clinical applicability of xenotransplantation. Here we show, using models involving T-cell-deficient athymic recipient mice, that rapidly induced, T-cell-independent xenoantibody production is mediated by marginal zone B lymphocytes and requires help from natural killer (NK) cells. This collaboration neither required NK-cell-mediated IFN-gamma production, nor NK-cell-mediated cytolytic killing of xenogeneic target cells. The T-cell-independent IgM xenoantibody response could be partially suppressed by CD40L blockade.

Induction and maintenance of T-dependent or T-independent xenotolerance by nonprimarily-vascularized skin or thymus grafts.

BACKGROUND: The success of clinical xenotransplantation will depend on induction of xenotolerance. We have previously shown that combined xenothymus and vascularized xenoheart transplantation under the coverage of a tolerizing regimen (TR) can induce and maintain full xenotolerance. Here, induction/maintenance of xenotolerance using nonprimarily-vascularized thymus and/or skin grafts was investigated. MATERIALS AND METHODS: Hamster skin or thymus or combined skin and thymus transplantation was performed in nude rat recipients with or without administering a TR (NK cell depletion, day -14; xenoantigen infusion, day -14; Leflunomide, day -14 through +14). Xenotolerance was confirmed by subsequent transplantation of a vascularized hamster heart, measurement of xenoantibody formation, or mixed lymphocyte reaction (MLR). RESULTS: Skin grafts were as effective as vascularized heart grafts to induce/maintain T-independent xenotolerance. Even without TR and despite being rejected themselves, xenoskin grafts lead to progressively developing xenononreactivity. Xenothymus transplantation induced xenotolerance in the T-dependent but not in the T-independent immune compartment, leading to rejection of subsequently transplanted hamster hearts by T-independent mechanisms (production of IgM but not IgG xenoantibodies (Xabs), presence of antihamster MLR nonresponsiveness). Combined skin and thymus xenotransplantation sensitized the T-cell compartment, leading to hyperacute rejection of subsequently transplanted hamster hearts. This was not the case when the skin grafts were transplanted late (2 months) after the thymus grafts. CONCLUSIONS: Xenogeneic skin and xenogeneic thymus grafts have opposite xenotolerance inducing capacities in the T-independent as compared to the T-dependent immune compartment. Thymus grafts induce and maintain T-dependent but not T-independent xenotolerance. Skin grafts alone induce T-independent xenotolerance but sensitize the T-cell compartment when transplanted concomitantly with thymus grafts.

Effects of a short course of leflunomide on T-independent B-lymphocyte xenoreactivity and on susceptibility of xenografts to acute or chronic rejection.

BACKGROUND: Leflunomide is a novel immunosuppressive agent with promising activity for xenotransplantation. It is not clear yet which mechanisms of action of leflunomide are responsible for that. METHODS: In a hamster-to-C57BL/6 nude mouse heart transplantation model, a 2-week course of leflunomide was used after transplantation or for pretreating donors. Nontolerant B lymphocytes were transferred to recipients after transplantation of first or second xenogeneic heart grafts that were transplanted with or without leflunomide treatment. RESULTS: Hamster xenogeneic hearts transplanted into athymic C57BL/6 nude mice receiving leflunomide did not induce immunoglobulin (Ig) M xenoantibodies (XAb) and survived without signs of chronic rejection. Second xenogeneic hearts transplanted 4 weeks after withdrawal of leflunomide survived without induction of XAb but developed chronic vascular lesions. After injection of naive B lymphocytes at 6 weeks after grafting a first or second hamster heart, only in the latter case were XAb induced. These were deposited in, and provoked acute rejection of, only the second grafts. Pretreatment of donors with leflunomide decreased the ex vivo xenoantibody deposition on the xenogeneic heart endothelia. CONCLUSIONS: A short posttransplant course of leflunomide induces T-independent B-lymphocyte xenotolerance. Leflunomide treatment also influences xenoantigen expression, as nontolerant B lymphocytes provoke IgM XAb formation and rejection of only second xenografts (transplanted without leflunomide) and not of first xenografts (transplanted with leflunomide treatment). The ex vivo experiments that show that XAb deposition is decreased in leflunomide-pretreated xenografts further confirm this. The latter may also explain the resistance of first and not second xenografts against chronic rejection.

Pathogenesis of autoimmunity after xenogeneic thymus transplantation.

Thymus transplantation is a promising strategy to induce xenotolerance, but may also induce an autoimmune syndrome (AIS). The pathogenesis of this AIS was explored using nude rats as recipients. Thymus grafts consisted of fetal hamster thymic tissue with or without mixing with fetal rat tissue such as thymus, thyroid, salivary gland, and heart. All hamster thymus recipients died of AIS within 2-3 mo. In most recipients of xenothymus mixed with rat tissues such as thymus, thyroid, and salivary gland, but not heart, AIS was prevented, indicating an insufficient presence of rat epithelial cell Ags within the xenothymus. AIS could be transferred to control nude rats by whole splenocytes or by splenocyte subpopulations such as CD3(+), CD3(-), and B lymphocytes, but not by non-T, non-B cells from AIS animals. This transfer could be suppressed by cotransferring either CD4(+) or CD8(+) lymphocytes from euthymic rats, but not by splenocytes from recipients of syngeneic or xenogeneic thymus mixed with rat tissue, indicating a defective generation of regulatory lymphocytes. As for CD4(+) regulatory cells this defect was probably qualitative, because the percentages of CD4(+)CD25(+) or CD4(+)CD45RC(low) populations were normal after xenothymus transplantation. As for the CD8(+) regulatory cells, the defect was quantitative, as CD8(+) cell levels always remained low. The latter was related to the nonvascularized nature of thymus grafts. In conclusion, AIS after xenothymus transplantation in nude rats is due to a combination of insufficient intrathymic presence of host-type epithelial cell Ags and a defective generation of regulatory T lymphocytes.