Ajoene inhibits the activation of human endothelial cells induced by porcine cells: implications for xenotransplantation

Abstract: Ajoene, is an organosulfur compound derived from garlic that strongly inhibit platelet aggregation, proliferation of human lymphocytes induced by phytohemagglutinin, and in general, blocks membrane-mediated signaling of cell activation. As a thrombotic microangiopathy frequently complicates procedures designed to induce pig-to-baboon chimerism by infusion of large amounts of pig progenitor cells in baboons, it was thought that ajoene might be useful to prevent such complication. For such purpose, we studied the effects of ajoene on the activation of human umbilical vein endothelial cells (HUVEC) induced by pig peripheral blood mononuclear cells (p-PBMC). Co-cultures of p-PBMC with HUVEC results in activation of the HUVEC as shown by over-expression of E-selectin and vascular cells adhesion molecule-1 (VCAM-1). Ajoene (25 μ m ) strongly inhibits HUVEC activation induced by tumor necrosis factor-α (TNF-α) or p-PBMC as shown by a down regulation of VCAM-1 and of E-selectin expression. After 5 or 8 h of pre-treatment with Ajoene, HUVEC incubated with TNF and p-PBMC showed an E-selectin or VCAM-1 expression, respectively, at levels similar to the positive control indicating that the inhibitory effect is transient. Ajoene at concentration of 25 μ m or lower did not affect HUVEC viability. Based on the finding that Ajoene has a strong, although transient, inhibitory effect on the activation of the endothelium induced by pig cells and its known anti-platelet activity, it is suggested that this garlic compound could be useful to prevent the development of microangiopathy and thrombotic disorders seen in primates infused with pig cells.     

ASGR1 expressed by porcine enriched liver sinusoidal endothelial cells mediates human platelet phagocytosis in vitro

Paris LL, Chihara RK, Reyes LM, Sidner RA, Estrada JL, Downey SM, Milgrom DA, Joseph Tector A, Burlak C. ASGR1 expressed by porcine enriched liver sinusoidal endothelial cells mediates human platelet phagocytosis in vitro. Xenotransplantation 2011; 18: 245–251. © 2011 John Wiley & Sons A/S. Abstract: Background: Porcine liver xenografts represent a potential solution to the organ shortage, but thrombocytopenia occurs within minutes to hours after xenotransplantation, preventing clinical application. Recently, it was discovered that porcine liver sinusoidal endothelial cells (LSEC) bind and phagocytose human platelets. We examined the role of ASGR1 in binding and removing human platelets by the pig liver endothelium. Methods: Primary porcine enriched LSEC (eLSEC) were characterized by flow cytometry, immunoblot, quantitative PCR, and immunohistochemistry using confocal microscopy. Phagocytosis inhibition assays using anti‐ASGR1 and an ASGR1 substrate were performed. ASGR1 was targeted for siRNA knockdown, and ASGR1‐reduced cells were tested for human platelet binding and phagocytosis. Results: ASGR1 is expressed by eLSEC. Human platelet binding and phagocytosis by porcine eLSEC was inhibited by asialofetuin, but not fetuin, suggesting an interaction with galactose β1‐4 N‐acetyl glucosamine. Anti‐ASGR1 antibodies inhibited human platelet binding in a dose‐dependent manner. Knockdown experiments using siRNA reduced ASGR1 expression in asynchronous primary eLSEC by 40%–80%. There was a 20% reduction in translated protein significantly correlated with a 21% decrease in human platelet binding. Conclusions: ASGR1 on porcine eLSEC mediates phagocytosis of xenogeneic platelets.     

Physiological role of sinusoidal endothelial cells and Kupffer cells and their implication in the pathogenesis of liver injury

We reviewed the morphological characteristics and physiological functions of hepatic sinusoidal endothelial cells (SECs) and Kupffer cells (KCs), both of which are major components of the hepatic sinusoid, and we showed the implication of these hepatic sinusoidal lining cells in the pathophysiology of the liver, based on our experimental studies. The most outstanding feature of SECs is that they are provided with numerous fenestrae, thereby allowing direct communication between the sinusoidal lumen and the space of Disse. Physiologically, SECs play a role in filtration function, endocytic function, and putative participation in the regulation of sinusoidal blood flow. As for KCs, they account for major portion of fixed macrophages in the entire body, and exhibit vigorous activity for phagocytosis, and produce many kinds of soluble mediators such as cytokines, prostanoids, oxygen radicals, and proteases. To determine whether these cells are implicated in pathophysiological processes in the liver we directed our attention to liver injury associated with sepsis and cold-preservation injury of liver tissue. In a septic rat model, we found that when KCs that included hepatic macrophages were activated, they released excess tissue-toxic mediators, probably leading to SEC damage. In the cold-preserved liver,we demonstrated that KCs were functionally activated and that the morphology of SECs was destroyed. When the liver was reperfused with plasma and a leucocyte suspension, hypercoagulability and increased leucocyte adherence occurred. In both experimental models, we demonstrated that KC blockade ameliorated the liver injury, and this was associated with the morphological improvement of SECs. Thus, we showed the pathogenetic implication of KCs and SECs, due possibly to microcirculatory disturbance in the hepatic sinusoid, and further emphasized the involvement of activated KCs in SEC impairment. Received for publication on Nov. 19, 1998; accepted on March 1, 1999     

Potential implications of ABO blood group for vascular rejection in pig to human kidney xenotransplantation

Abstract: Background: A substantial hurdle for successful xenotransplantation is to negate the effect of xenoreactive natural antibodies [mainly Galα1–3Galβ1–4GlcNAc (α‐Gal) specific] that cause hyperacute xenograft rejection. Galα1–3Gal molecules (α‐Gal) have close structural homology with human ABO blood groups and therefore an individual's blood group might influence the formation of α‐Gal specific antibodies. Genetic heterogeneity controlling α‐Gal specific antibody formation could have important implications for future pig to human xenotransplantation clinical trials. We have investigated the relationship between ABO blood group and immunoglobulin M (IgM) and immunoglobulin G (IgG) α‐Gal specific antibody titres in sera obtained from renal dialysis patients and healthy blood donors. Methods: Serially diluted sera (n = 166) obtained from renal dialysis patients awaiting kidney transplantation (n = 116) and healthy blood donors (n = 50) were tested for IgM and IgG α‐Gal antibodies using an enzyme‐linked immunosorbent assay (ELISA) specific for α‐Gal. The study cohort comprised 62, 48, 36 and 20 sera obtained from blood group O, A, B and AB individuals, respectively. Reciprocal α‐Gal specific antibody titres were calculated from ELISA titration curves and stratified by individual blood group. Results: No significant heterogeneity was found in IgM α‐Gal specific antibody titres across ABO blood groups. In contrast, marked heterogeneity was observed in IgG α‐Gal specific antibody titres when stratified by blood group. IgG α‐Gal specific antibody titres were higher in sera obtained from blood group O renal dialysis patients [median titre 40, interquartile range (IQR) 14 to 72], compared with blood group A (median titre 18, IQR 7 to 54, P = 0.05), blood group B (median titre 6, IQR 0 to 15, P < 0.001) and blood group AB patients (median titre 3.5, IQR 0 to 16, P = 0.002). A similar correlation was found for IgG α‐Gal specific antibody titres in sera obtained from healthy blood donors with median titres of 20 (IQR 12 to 34), 37 (10 to 91), 9 (0 to 20), and 5.5 (0 to 12) in blood groups O, A, B and AB individuals, respectively. There was a strong interrelationship between α‐Gal specific antibody class and blood group, with both IgM and IgG α‐Gal specific antibodies found in 84% of the blood group O sera, 73% of blood group A sera, 50% of blood group B sera and 40% of blood group AB sera (P < 0.001). In a subgroup of 39 renal dialysis patients, IgM and IgG α‐Gal specific antibody titres were measured in two serum samples obtained at different time‐points (median time interval 581 days, range 42 to 4414), and showed a high degree of stability (correlation coefficient 0.88 and 0.90 for IgM and IgG, respectively). Conclusion: IgG α‐Gal specific antibody titres are significantly higher in the sera of blood group O and A renal dialysis patients and healthy individuals compared with blood groups B and AB. These data indicate that future clinical trials of pig to human xenotransplantation may be more problematic for non‐blood group B patients who are likely to have high levels of IgG α‐Gal specific antibodies that are associated with acute vascular rejection.     

Compatibility of porcine and human interleukin 2: implications for xenotransplantation

BACKGROUND: Xenotransplantation provides a possible solution to the severe shortage of allogeneic organ donors. The pig, which shares many physiological similarities with humans, makes it an optimal species for preclinical experimentation and clinical applications. Interleukin 2 (IL2) is a potent growth factor secreted primarily by T helper lymphocytes and it is vital to the cellular expansion required for a productive immune response and the development and peripheral expansion of CD4+CD25+ regulatory T cells. Therefore, it is essential to understand of the compatibility of IL2 between pigs and humans. METHODS: We first compared the cDNA and protein sequences and the crystal structures of human and porcine IL2 and IL2 receptors, respectively. The effect of IL2 to induce T cell proliferation was determined by 3H-thymidine incorporation and cell cycle detection. RESULTS: Porcine IL2 induced very limited proliferation of human lymphocytes while it functioned well on porcine lymphocytes. Human IL2 had remarkably reduced effects on porcine lymphocytes whereas it worked well on human lymphocytes. CONCLUSION: Our present study showed that the interaction of IL2 and IL2R across species might have defects. Together with the wide physiological functions of IL2, our data indicated that physiological disorders could be caused by the poor function of xenogeneic donor IL2 on host cells in full hematopoietic chimera. Our data suggested an additional potential advantage for the mixed xenogeneic chimeras.     

A review of pig liver xenotransplantation: Current problems and recent progress

Pig liver xenotransplantation appears to be more perplexing when compared to heart or kidney xenotransplantation, even though great progress has been achieved. The relevant molecular mechanisms involved in xenogeneic rejection, including coagulopathy, and particularly thrombocytopenia, are complex, and need to be systematically investigated. The deletion of expression of Gal antigens in the liver graft highlights the injurious impact of nonGal antigens, which continue to induce humoral rejection. Innate immunity, particularly mediated by macrophages and natural killer cells, interplays with inflammation and coagulation disorders. Kupffer cells and liver sinusoidal endothelial cells (LSECs) together mediate leukocyte, erythrocyte, and platelet sequestration and phagocytosis, which can be exacerbated by increased cytokine production, cell desialylation, and interspecies incompatibilities. The coagulation cascade is activated by release of tissue factor which can be dependent or independent of the xenoreactive immune response. Depletion of endothelial anticoagulants and anti‐platelet capacity amplify coagulation activation, and interspecies incompatibilities of coagulation‐regulatory proteins facilitate dysregulation. LSECs involved in platelet phagocytosis and transcytosis, coupled with hepatocyte‐mediated degradation, are responsible for thrombocytopenia. Adaptive immunity could also be problematic in long‐term liver graft survival. Currently, relevant evidence and study results of various genetic modifications to the pig donor need to be fully determined, with the aim of identifying the ideal transgene combination for pig liver xenotransplantation. We believe that clinical trials of pig liver xenotransplantation should initially be considered as a bridge to allotransplantation.     

非协调性异种肝移植中移植肝超急性排斥反应的病理形态学表现

目的:了解豚鼠至大鼠异种肝移植动物模型中移植肝病理学表现。方法:利用血管套技术和显微外科技术进行了20例豚鼠至大鼠肝移植。观察了受体存活时间和移植肝HE染色情况、电子显微镜下表现和荧光染色表现。用TUNEL法检测了移植肝细胞凋亡情况。结果:受体存活时间平均为(135.10±46.12)min,组织学表现为肝小叶结构存在,但肝细胞发生弥漫性水样变性,中央静脉和小叶间静脉淤血;电子显微镜下见肝细胞内糖原颗粒消失,细胞器水肿,近血窦处肝细胞膜破坏;IgM和IgG染色主要位于肝脏血管内皮细胞表面和肝血窦内。细胞凋亡指数为(0.80±0.31)％。结论:首次建立了豚鼠至大鼠异种肝移植模型,观察了超急性排斥反应。     

On the intersections of basic and applied research in xenotransplantation

I am very grateful to the Council and members of the International Xenotransplantation Association for this Honorary Membership. In accepting this prestigious award, I pay tribute to my mentors Antonio Oriol i Anguera, Carlos Martinez, Robert A. Good, and Hans Müller-Eberhard for their guidance and friendship as I was beginning my travels in biomedical research. I also thank the many gifted collaborators, students, and technical personnel, as well as the agencies and taxpayers, who funded our research and made our scientific contributions possible. Here I briefly mention some of these contributions, including early work on the immunobiology of the thymus, my short incursion in the immunology of Chagas disease, and what have been the dominant themes of my career: the mechanisms of complement injury, the role of complement in pathophysiology, and induction of cytoprotection in the vascular endothelium. I emphasize our contributions on the role of complement as related to understanding and overcoming xenograft injury, a work that has been personally very rewarding. Now it is exciting to see that the field of xenotransplantation research is moving forward vigorously, a time of great optimism suggesting that many potential clinical applications of xenotransplantation will come to fruition in the near future.     

Prostaglandin E1 (PGE1) attenuates vasoconstriction induced by PGE2, PGD2 and phorbol myristate acetate in the perfused rat liver

It has been shown that prostaglandins (PGs) produced by Kupffer and endothelial cells play an important role in mediating physiological responses to various immunological stimuli. We studied the effect of prostaglandin E₁ (PGE₁) on the hemodynamic and metabolic changes induced by prostaglandin E₂ (PGE₂), D₂ (PGD₂) and phorbol 12-myristate 13-acetate (PMA), a potent inducer of PGs in the isolated rat liver perfused with Krebs-Ringer-bicarbonate (KRB) solution at a constant pressure of 12cmH₂O. The liver was taken from overnight-fasted male Sprague-Dawley rats weighing 260 to 310g. Both PGE₂ and PGD₂ significantly decreased hepatic flow when their initial concentration was elevated to micromolar range. Although 1 × 10^–6M of PGE₁ did not have a major effect on hepatic flow, it significantly attenuated the declines of hepatic flow produced by 4 × 10^–6M of PGE₂ and PGD₂. However, none of PGs tested influenced glucose and lactate concentrations in the medium. Continuous infusion of PGE₁ into the medium at a rate of 5µg·min^–1 significantly diminished the decreases in hepatic flow and oxygen consumption induced by 2 × 10^–8M of PMA. These results suggest that administration of PGE₁ may preserve hepatic blood flow by modifying the intrahepatic regulatory mechanism involving the activation of Kupffer and endothelial cells.(Inaba H, Araki M, Numai T, et al.: Prostaglandin E₁ (PGE₁) attenuates vasoconstriction induced by PGE₂, PGD₂ and phorbol myristate acetate in the perfused rat liver. J Anesth 7: 56–65, 1993)     

Monomorphic and polymorphic carbohydrate antigens on pig tissues: implications for organ xenotransplantation in the pig-to-human model

The existence of the Gal epitope in 137 pigs belonging to 23 different breeds suggests that this antigen is either monomorphic or occurs at a high incidence in the porcine species. Its histological location at the surface of pig vascular endothelial cells makes it a target for human natural anti-Gal antibodies and complement, which may be responsible for the hyperacute vascular rejection of transplanted pig organs. The precursor carbohydrate chain (N-acetyllactosamine) and NeuAc-substituted epitopes are also exposed at the surface of pig vascular endothelium and were found in all pigs in this study. However, humans also have these two epitopes on vascular endothelium and, consequently, have not made natural antibodies against these carbohydrate antigens. Therefore, these two pig epitopes cannot be the main target of the hyperacute vascular rejection process. Three pig phenotpyes-A+(51%), A:H+(38%), and A-H-I+(11%) were identified among 37 Large-white pigs by the presence of polymorphic A, H, and I carbohydrate antigens on the brush border of the surface epithelium of small intestine. These antigens were also present in other exocrine secretions but were not detected on vascular endothelium of the same pigs, suggesting that they are not involved in the hyperacute vascular rejection, although the pig A tissue antigen can induce an immune response in 0 or B blood group recipients. Once the problem of the initial hyperacute vascular rejection directed against the Gal epitope is overcome, typing donor pigs for A, H, and I, as well as for the protein swine leukocyte antigens (SLA) and other pig antigens, may help in elucidating antigens involved in acute or chronic xenograft rejection.     

Role of Kupffer cells in the survival after rat liver transplantation with long portal vein clamping times

Applying the orthotopic rat liver transplantation (ORLT) model, postoperative survival has been shown to be mainly dependent on the portal vein clamping time (PVCT). It was hypothesized that prolonged intestinal congestion was responsible for the activation of Kupffer cells (KC) with overproduction of TNF, secondary to splanchnic endotoxin accumulation and release on reperfusion. The role of KCs was directly investigated in the context of long PVCTs by eliminating them (using liposome-encapsulated dichloromethylene diphosphonate), by preventing their activation (using a calcium channel blocker, nisoldipine) and by inhibiting TNF production (using thalidomide). Livers from different groups of rats were transplanted following 24-h cold preservation in the UW solution with long PVCTs (from 18–21 min). KCs depletion, preservation with nisoldipine and pretreatment with thalidomide significantly improved survival in conditions using long PVCTs. KC depletion and nisoldipine preservation had no effect on liver enzymes or pathological findings while lung injury was significantly improved. The present data confirm that, in the context of ORLT with long PVCTs, KCs are directly responsible for the systemic endotoxin-like shock syndrome and their effect is mediated through overproduction of TNF. Received: 30 August 1999 Revised: 18 May 2000 Accepted: 12 September 2000     

Experimental discordant hepatic xenotransplantation in the recipient with liver failure: implications for clinical bridging trials

BACKGROUND: Clinical xenotransplantation might start with bridge-to-bridge trials. Situations where hyperacute rejection is avoided would provide opportunities for the initiation of bridging trials. Patients with liver failure have a diminished capacity to initiate antibody and complement-induced injury of xenogeneic endothelium. Hyperacute rejection of a liver xenograft manifests as a coagulopathy. We examined the ability of a recipient with liver failure to hyperacutely reject a liver xenograft in the dog-to-pig model in the immediate postoperative period. STUDY DESIGN: Liver failure in pigs was induced with galactosamine. Canine livers were transplanted into pigs with liver failure and into healthy pigs. The postoperative course was monitored for 1 hour for histologic changes in the xenograft, changes in platelet counts, and whole blood clotting with Sonoclot analysis. In vitro assays with pig serum and canine hepatic sinusoidal endothelial cells were used to assess the effect of liver failure on serum cytotoxicity and xenoreactive antibody levels. RESULTS: All untreated pig recipients of liver xenografts died from a coagulopathy. Recipients with liver failure manifested no signs of coagulopathy, and had minimal change in platelet counts or Sonoclot (Sienco Inc., Morrison, CO) tracings. Liver xenograft biopsies from recipients with liver failure showed no evidence of the tissue injury that characterized the biopsies of control recipients. Serum from pigs was less cytotoxic to the canine hepatic sinusoidal endothelium after induction of liver failure. The xenoreactive antibody levels and repertoire were similar in the pig serum before and after liver failure was induced. CH50 (total complement) levels were diminished in pigs after the induction of liver failure. CONCLUSIONS: Liver xenotransplantation used in bridging trials in recipients with liver failure might not face the barrier of hyperacute rejection.     

A polymerase chain reaction-based protocol for the detection of transmission of pig endogenous retroviruses in pig to human xenotransplantation

BACKGROUND: Xenotransplantation of pig organs and tissues to humans bears the risk of infection of immunosuppressed recipients by porcine endogenous retrovirus (PERV) released from the transplanted tissue. However, when diagnosing potential PERV transmission, it is essential to exclude microchimerism, i.e., persisting pig cells in analyzed bioptic material of xenotransplanted patients, which give rise to false positive PERV signals. Polymerase chain reaction (PCR) is so far the only suitable method to diagnose a cross-species transfer of PERV, but the exclusion of microchimerism might be a serious problem because most of the presently employed primer pairs detect PERV sequences with higher sensitivity than primers used for the detection of contaminating pig sequences. METHODS: We designed and evaluated a novel and improved primer set for detection of pig sequences as well as complementing positive control primers on the basis of mitochondrial cytochrome B, an approved marker for phylogenetic studies. We further established primer pairs derived from the long terminal repeat/leader region of PERV isolated from a Duroc German Landrace cross-bred pig and tested their sensitivity in comparison with known PERV- and pig-specific PCR markers. RESULTS: In standard PCR assays, the new cytochrome B-derived primers are at least 10 times more sensitive than the presently used PERV retroviral polymerase gene and mammalian beta-actin primers. When tested in a tissue culture infection model, PERV transmission to human 293 cells can be unambiguously demonstrated, even in the presence of up to 10% pig cells. One of the primer combinations derived from the PERV DuxDL3791 long terminal repeat/leader region amplifies with even lower sensitivity than primers detecting porcine beta-globin, thus permitting the exclusion of microchimerism also via chromosomal loci. CONCLUSIONS: The availability of the new PCR markers allows the proposal of a rigorous setup for the routine detection of PERV transmission after xenotransplantation.     

Antibodies to human adhesion molecules and von Willebrand factor: In vitro cross-species reactivity in the xenotransplantation setting

Abstract: Endothelial cell activation is thought to play an important role in xenograft rejection through cell retraction and expression of pro-coagulant and pro-inflammatory factors. Identification of antibodies recognizing porcine endothelial molecules would be useful to study and manipulate the inflammatory response to a xenograft. The aim of this study was to investigate the cross-reactivity of antibodies directed against human adhesion molecules and von Willebrand factor (vWF). Binding of monoclonal antibodies (mAbs) directed against human CD31, CD44, CD49, CD54, CD62E, CD102, and CD106 was evaluated on resting and activated endothelial cells from human and pig by flow cytometry. Among 30 antibodies tested, 4 were shown to react with pig cells. Two of them, directed against human CD62E (E-selectin) and rabbit CD 106 (VCAM-1) reacted strongly with activated and/or resting pig cells, whereas two others, directed to human CD31 (PECAM) and CD44 (H-CAM), bound weakly to pig cells. In addition, we analyzed the cross-reactivity of five polyclonal or monoclonal antibodies to human or pig vWF with human, baboon, rhesus, pig, and rat vWF. Binding of antibodies was tested by ELISA by using platelet lysates as source of vWF from the different species. Four anti-human or porcine vWF antibodies exhibited a broad reactivity with vWF from all species, whereas one anti-human vWF antibody was specific for primate vWF. In this study, we identified a small number of cross-reacting antibodies that may prove useful to study in vitro and in vivo xenogeneic responses. However, the weak antibody cross-reactivity observed with most porcine molecules points out the necessity of producing species-specific antibodies to study the immune response to xenografts or for use as specific immunosuppressive therapeutic reagents.     

The fate of recipient-derived hepatocytes in sex-mismatched liver allograft following liver transplantation

BACKGROUND: 'Bone marrow-derived stem cells' have attracted great attention as potential candidates for liver-directed gene therapy and as a tool for regenerative medicine. However, the fate of these cells is not well-known. The aim of this present study was to investigate the fate of 'recipient-derived repopulated hepatocytes' in sex-mismatched liver allografts in individuals following liver transplantation during systematic longitudinally performed liver biopsies. METHODS: Paraffin-embedded sex-mismatched liver biopsy samples of nine recipients (male/female ratio 5/4; mean age: 39.7 yr) were reviewed. Double labeling with immunohistochemistry for hepatocytes and recipient-specific bone marrow-derived cells and fluorescence in-situ hybridization for visualizing X and Y chromosomes were performed. These slides were examined systematically using an image analyzer system (Olympus microscope; Cyto-Vision, Applied Imaging, Biosciences Centre, Newcastle, UK). Only cells with two nuclear spots were considered for interpretation. RESULTS: The mean times from transplantation to first biopsy and between the first and the second biopsies were 5.9 and 20.9 months respectively. The proportion of recipient-derived repopulated hepatocytes was significantly decreased in the late biopsies when compared with the early biopsies (p = 0.001). All nine samples of the first biopsies had demonstrated recipient-derived hepatocyte repopulation, with a mean of 2.0%, whereas only seven of nine samples of the second biopsies had demonstrated recipient-derived hepatocyte repopulation with a low mean of 0.5% (p = 0.001). CONCLUSION: Based on these results, we suggest that 'recipient-specific bone marrow-derived hepatocyte repopulation' in liver allograft during tissue injury is a relatively early event.     

异种反应性天然抗体在豚鼠至大鼠肝移植中的作用

目的　研究异种反应性天然抗体 (XNA)在豚鼠至大鼠异种肝移植超急性排斥反应(HAR)中的作用。方法　将实验鼠随机分成A、B、C、D组 ,每组 2 0只 ,分别为对照组、术前输注豚鼠肝细胞 (HC)组、术前连续肌注山地明 (CsA)组和术前输注HC合用CsA组。采用流式细胞仪和免疫组织化学方法检测受体体内XNA含量 ,观察了受体存活时间和移植肝组织学改变。结果　移植肝组织发生了HAR。受体体内存在XNA ,以IgM为主。术前输注HC使受体体内的抗体明显升高 ,A组IgM(单位 :平均荧光强度 ,下同 )为 74.58± 31 .75 ,B组为 40 6 .42± 1 0 8.0 2 (P <0 .0 1 ) ,而使用CsA能预防抗体爆发反应 ,C组为 48.82± 1 1 .0 4 (同B组比较 ,P <0 .0 1 )。术前输注豚鼠肝细胞合并使用CsA能延长受体存活时间 ,A组为 (1 2 4 .1 0± 33 .42 )min,D组为 (1 83 .70± 2 6 .85)min(P <0 .0 1 )。移植肝表现为肝细胞水样变性 ,肝血窦和血管扩张瘀血 ,但小叶结构完整。结论在豚鼠至大鼠异种肝移植中发生的HAR是一种强烈的免疫反应 ,其中XNA所起作用有限 ;术前输注豚鼠肝细胞合并使用CsA能延长受体存活时间     

供体骨髓间充质干细胞干预非协调性异种肝移植免疫排斥反应的实验研究

目的研究骨髓间充质干细胞(MSCs)对非协调性异种肝移植排斥反应的免疫干预作用。方法密度梯度离心法分离培养豚鼠MSCs,流式细胞仪检测其膜表面CD34、CD45、CD44和CD29的表达,脂肪细胞诱导液培养诱导MSCs向脂肪细胞分化。豚鼠MSCs输注大鼠后检测不同时相免疫球蛋白IgG、IgM、IgA以及补体C3、C4的浓度变化。豚鼠和大鼠各30只随机分为3组,所有大鼠受体经环磷酰胺预处理,A组为MSCs输注组;B组为生理盐水输注组;C组为地塞米松输注组。各组行豚鼠大鼠原位肝脏移植手术,观察受体的存活情况及排斥反应情况。结果MSCs膜表面CD34阴性、CD45阴性、CD44阳性、CD29阳性。脂肪细胞诱导液培养后细胞内出现脂滴沉着。MSCs细胞悬液输注受体后各个时相的免疫球蛋白IgG、IgM和补体C3较输注前有显著降低(P<0.01),而IgA和补体C4与输注前相比无明显变化(P>0.05),输注后的免疫球蛋白IgG、IgM和补体C3各个时相点之间的浓度变化无显著差异(P>0.05)。肝脏移植模型A组受体存活时间为(431±27)min,较B组和C组的存活时间[(148±16)min、(141±22)min]显著延长(P<0.01)。A组受体超急性排斥反应发生较迟,程度轻。结论MSCs的鉴定可根据细胞形态,膜表面标志物和分化能力来确定,MSCs可干预异种肝脏移植的超急性排斥反应的发生。     

The effects of the elimination of Kupffer cells in the isolated perfused liver from non‐heart‐beating rat

Abstract We examined the effect of elimination of Kupffer cells on the sinusoidal microcirculation in graft harvested from non‐heart‐beating donors (NHBD), focusing on the arachidonic acid cascade and cytokines. Cardiac arrest was induced by thoracotomy. Livers were harvested 30 min after thoracotomy and perfused by Krebs‐Henseleit bicarbonate buffer for 60 min after 6 h cold preservation. For the elimination of Kupffer cells, rats were pretreated liposome‐encapsulated dichloromethylene diphosphonate (KE group). Eicosanoids (TXB₂, 6‐keto‐PGF_1α, LTB₄) and cytokines (TNFα, IL‐1β) in the perfusate were measured. Histological examination was also carried out. In the KE group, the value of TXB₂ was suppressed completely and cytokines were reduced, and sinusoidal structures and hepatocytes were well protected. These results indicated that the elimination of Kupffer cells improved sinusoidal microcirculation in NHBD and liver transplantation using grafts from NHBD could be made to succeed by modulation of Kupffer cells.