Abnormality of coagulation and fibrinolysis functions of endothelial cells induced by activated complement alternative pathway and intervention / 中国药理学通报

Aim To investigate the change of coagulation and fibrinolysis functions of human microvascular endothelial cells (HMEC) induced by activated complement alternative pathway and effect of pyrrolidine-dithiocarbamate (PDTC) and resveratrol (Res) on intervention.Methods Normal human serum was activated by cobra venom factor (CVF).After exposure of HMEC to activated complement for various time points, supernatant was removed and assayed for activities of hydrolysing chromogenic substrate and affecting activated partial thromboplastin time (APTT) and prothrombin time (PT).The cells exposed to activated complement were collected and washed, and then the cell suspension was assayed for activity of affecting coagulation function of normal plasma.Lastly, the coagulation and fibrinolysis functions of HMEC pretreated with PDTC and Res were also investigated after HMEC was exposed to activated complement alternative pathway.Results The hydrolysis activity of chromogenic substrate of supernatant was up-regulated significantly after HMEC exposed to activated complement alternative pathway.The supernatant induced APTT decreased significantly, and also shortened PT.The cell suspension of various time points induced APTT decreased significantly, and also shortened PT by suspension of 6 h time point.PDTC and Res failed to inhibit the up-regulation of the chromogenic hydrolysis activity, but Res showed significant intervention on decrease of APTT, and PDTC had better effect on inhibiting the decrease of PT than that of Res.Conclusion Activated complement alternative pathway can induce abnormality of coagulation and fibrinolysis functions of HMEC, and PDTC and Res can affect this change.

Effect of chlorogenic acid,caffeic acid,and ferulic acid on inhibition of inflammatory response of HMECs induced by activated complement alternative pathway / 中国药理学通报

Aim To investigate the effect of chlorogen-ic acid,caffeic acid,and ferulic acid on expression of molecules related with inflammatory response of HMECs induced by activated complement alternative pathway.Methods CVF was used to activate the al-ternative pathway of serum complement.After exposure of HMECs to activate complement for various times, supernatant of cell culture was removed and assayed for content of ICAM-1,IL-6,IL-8,t-PA,and PAI-1 u-sing ELISA kits.The expression of the above mole-cules induced by activated complement was measured after HMECs were pre-treated with 50,100,250 μmol ·L-1 of CGA,CA,and FA.Results After HMECs were exposed to the product of the activated comple-ment alternative pathway,the expression of ICAM-1 , IL-6,IL-8,t-PA,and PAI-1 was up-regulated.The expression of ICAM-1,IL-6,IL-8,t-PA,and PAI-1 was down-regulated by various concentrations of CGA, CA,and FA.ICAM-1 and IL-8 were inhibited most significantly in all molecules mentioned above.CA ex-hibited the best intervention effect,followed by FA. Conclusion Certain concentration of CGA,CA,and FA can inhibit the expression of ICAM-1,IL-6,IL-8, t-PA,and PAI-1 in HMECs induced by the activation of the alternative complement pathway,indicating that CGA,CA,and FA can inhibit inflammatory response of HMECs.

Inflammatory mechanism of acute lung injury in mice induced by activation of complement alternative pathway / 中国药理学通报

Aim To study the development of acute lung inflammation in mice induced by activation of the complement alternative pathway and the changes of the related indicators, and to provide an ideal pathological model of acute lung inflammation in mice for drug screening and intervention. Methods Cobra venom factor( CVF) was used to activate complement alterna-tive pathway of SPF Kunming mice by intravenous injection. According to different sampling time, the mice were divided into 15 min, 30 min, 1 h, 2 h, 6 h group, and the parallel PBS control groups were set at the same time. Lung coefficient, lung water content, myeloperoxidase ( MPO ) activity, BALF cell number and protein content were tested. The pathological changes of lung tissue were observed by HE staining. The concentration of IL-6 , TNF-α, P-selectin and ICAM-1 in bronchoalveolar lavage fluid ( BALF ) and serum were determined by ELISA. Results CVF caused pulmonary inflammatory cell infiltration in mice obviously. Compared with PBS groups, MPO activity of lung tissue, BALF cell and the protein concentration were significantly increased. The contents of IL-6, TNF-α, P-selectin in BALF and serum were in-creased, and the content of ICAM-1 in serum was also increased. The content of P-selectin in BALF reached the first peak at 30 min point, the content of IL-6 and TNF-α in BALF reached the first peak at 1 h point, but the indicators had no further changes at 2 h point, and all the indicators rose again at 6 h point. The lev-els of IL-6 and TNF-α in serum reached peak at 1 h point,then the content showed lower levels at the sub-sequent time points. The levels of P-selectin and ICAM-1 in serum increased along the time. Lung coef-ficient, lung water content and ICAM-1 of the BALF showed no significant alteration. Conclusion The ac-tivation of the complement alternative pathway can lead to acute lung inflammation in mice and the inflammato-ry response is the most obvious at 30 min to 1 h. The study could provide an ideal pathological model of a-cute lung inflammation in mice for drug screening and intervention.

Effect of Protobothrops mucrosquamatus venom and its fractions on bIood coaguIation / 中国药理学与毒理学杂志

OBJECTIVE To investigate the effect of Protobothrops mucrosquamatus venom (PMV) and its fractions on functions of the circulatory system in vitro in order to better understand its toxicity mechanism. METHODS PMV was isolated to three fractions FⅠ, FⅡ and FⅢ with a different molecular mass range by Sephadex G-75 gel filtration chromatography. Platelet rich plasma was adjusted to 3×1011 L-1 by platelet poor plasma. Platelet suspension was incubated with PMV and its fractions 0.03 g.L-1 for 5 min, respectively, and platelet aggregation was determined on an LBY-NJ4 aggregometer. PMV and its fractions 0.05 g.L-1 were preincubated with plasminogen 0.1 U.L-1 for 10 min before chromogenic substrate cleavage activity was measured by endpoint and enzyme kinetics determination. PMV and its fractions 1.0 g.L-1 were incubated with rat plasma for 5 or 30 min, and thrombin time (TT), activated partial thromboplastin time (APTT), prothrombin time (PT) and fibrinogen (FlB) content were assayed. The microvascular endothelial cells were exposed to PMV and its fractions 10, 50 and 250 mg.L-1 , respectively, for 24 h, while the morphological change was observed using an inverted phase contrast microscope, and the cell viability was determined by MTT method. PMV and its fractions were incubated with guinea pig red blood cell suspension in the presence or absence of lecithin for different time, and hemolysis was measured. RESULTS Compared with normal control, platelet aggregation rate was significantly increased by PMV and FⅠ (>71 ku)〔(12.4±4.1)%,(61.0±5.8)% and (56.9±5.9)%〕(P<0.01). PMV and FⅡ (18-37 ku) significantly hydrolyzed chromogenic substrate S-2251(P<0.01). PMV and FⅠ caused plasma coagulation. Compared with normal control, FⅡ and FⅢ (<10 ku) remarkably prolonged TT, APTT and PT( P<0.01). Morphological observation revealed that PMV, FⅠ and FⅡdetached the adherent cells. Compared with normal control group, PMV, F Ⅰ and F Ⅱ inhibited cell viability, and the survival rate of the cells decreased to (56.8±3.6)%,(71.6±3.8)% and(58.2±5.5)%, respectively. PMV and FⅡ slowly caused slight hemolysis in absence of lecithin. PMV and FⅡ caused significant hemolysis in the presence of lecithin, and the hemolytic rate increased to (81.0±4.0)% and (81.0±1.0)%( P <0.01) in 0.5 min, respectively, compared with (17.7±1.0)% of the control group. CONCLUSION PMV possesses different activities that affect the functions of the circulatory system in vitro, and the fractions play different roles in toxicity mechanisms.

Effect of three chemical molecules on adhesion molecules expression in HMECs induced by activated complement alternative pathway / 中国药理学通报

Aim To investigate the effect of resveratrol ( Res) , PDTC and AG490 on adhesion molecules ex-pression induced by product of activated complement alternative pathway on human microvascular endothelial cells ( HMECs) and the possible mechanisms. Meth-ods HMECs were exposed to the product of comple-ment alternative pathway activation, then the superna-tant was removed to detect the concentration of malond-ialdehyde ( MDA ) with TBA method. ELISA method was used to detect the expression of soluble ICAM-1 , VCAM-1 ( and E-selectin) in the culture supernatant. Res, PDTC and AG490 with different concentrations were used to determine their effect on cell oxidation level and adhesion molecules expression. The phospho-rylation of NF-κB p65 was detected by Western blot, and the intervention of Res, PDTC and AG490 was as-sayed by the same way. Results The activation of complements alternative pathway resulted in the phos-phorylation of NF-κB p65 , and increased the concen-tration of MDA and up-regulated the expression of ICAM-1, VCAM-1 and E-selectin. Res reduced the concentration of MDA. Res, PDTC and AG490 inhibi-ted the phosphorylation of NF-κB p65 . Res and PDTC showed similar inhibition on expression of ICAM-1 and VCAM-1 , while exhibiting little effect on expression of E-selectin, and AG490 significantly inhibited the ex-pression of the above adhesion molecules. Conclusions Res, PDTC and AG490 could inhibit the expression of adhesion molecules induced by activated complement alternative pathway, the inhibition of NF-κB pathway activation was involved in their mechanism, and JAK2 may be a more important intervention target in regula-ting adhesion molecule expression.

Expression of coagulation-and fibrinolysis-related molecules of endothelial cells induced by activated complement alternative pathway and intervention / 中国药理学通报

Aim To investigate the change of molecu-lar expression related to coagulation and fibrinolysis in human microvascular endothelial cells ( HMEC ) in-duced by activated complement alternative pathway and effect of pyrrolidine dithiocarbamate ( PDTC ) and res-veratrol on intervention. Methods Normal human se-rum was activated by cobra venom factor ( CVF) . After exposure of HMEC to activated complement for various times, supernatant was removed and assayed for ex-pressions of P-selectin, VWF, t-PA, PAI-1, TF, TM, and NO by using reagent kits. The expressions of the above molecules in HMEC pretreated with PDTC and resveratrol were also investigated. Results P-selectin and VWF were rapidly released by endothelial cells and the expression reached the peak at the time point of 15 min. The expressions of t-PA, PAI-1, and TF were continuously upregulated, whereas NO and TM were decreased. PDTC and resveratrol inhibited the upregulation of P-selectin, VWF, t-PA, PAI-1 and TF, and intervened the downregulation of NO. Res-veratrol further downregulated the expression of TM. Conclusion Activated complement alternative path-way can influence the expression of molecules related to coagulation and fibrinolysis in HMEC, and PDTC and resveratrol can affect this change.

Atorvastatin attenuates activation and injury of human microvascular endothelial cells induced by oxidized low density lipoprotein / 中国药理学通报

Aim To investigate the effects of atorvas-tatin ( ATV) on activation and injury of microvascular endothelial cells induced by oxidized low density lipo-protein ( ox-LDL) . Methods Cultured human micro-vascular endothelial cells were pre-incubated with ATV for 24 h prior to exposure of endothelial cells to ox-LDL. After exposure of endothelial cells to ox-LDL, the cell viability was measured by MTT method, LDH in supernatants was determined by enzyme activity as-say kit, ICAM-1 in supernatants was assayed by using ELISA method, phosphorylation of NF-κB p65 was de-tected by western blot analysis, transcriptional activity of NF-κB signal pathway was measured by employing dual-luciferase reporter assay system. Results Hu-man microvascular endothelial cells were activated and injured by ox-LDL. Inhibition of the cell viability, re-lease of LDH, expression of ICAM-1, phosphorylation of NF-κB p65 , and up-regulated transcriptional activity of NF-κB induced by ox-LDL were attenuated by ATV. Conclusion ATV can significantly inhibit the activa-tion and injury of human microvascular endothelial cells induced by ox-LDL, and that may be related to inhibition of phosphorylation and transcriptional activity of NF-κB.

A new oligosaccharide from Periploca calophylla / 中国中药杂志

<p><b>OBJECTIVE</b>To study the chemical constituents of Periploca calophylla.</p><p><b>METHOD</b>Various chromatographic techniques were used to isolate the constituents, and their structures were identified by spectral and chemical methods.</p><p><b>RESULT</b>Two oligosaccharides were isolated from the chloroform part of P. calophylla and their structures were identified as 4-O-acetyl-beta-cymaropyranosyl (1-->4)-O-beta-D-cymaropyranosyl(1-->4)-O-beta-D-canaropyranosyl (1-->4)-O-beta-D-cymaropyranosy(1-->4)-O-oleandronic acid-delta-lactone(1), and perisaccharide B (2).</p><p><b>CONCLUSION</b>Compound 1 is a new compound. Compound 2 is reported for the first time from this plant.</p>

Alkaloids in stems and leaves of Stephania cepharantha / 中国中药杂志

<p><b>OBJECTIVE</b>To study the alkaloids in the stems and leaves of Stephania cepharantha.</p><p><b>METHOD</b>The dried stems and leaves of S. cepharantha were percolated with 95% ethanol and the solvent was removed by rotary evaporation to give a concentrate, and the concentrate was extracted by petroleum ether and chloroform. Column chromatograghy on MCI CHP 20P, silica gel, Rp-18, Sephadex LH-20 and polyamide were applied for the isolation and purification of the chloroform fraction. The structures were elucidated by their physicochemical properties and spectral data.</p><p><b>RESULT</b>Eleven alkaloids were obtained and identified as lysicamine (1), tetrahadropalmatine (2), palmatine (3), isocorydione (4), corydalmine (5), corypalmine (6), sinoracutine (7), sinoacutine (8), cepharamine (9), isocorydine (10) and corydine (11).</p><p><b>CONCLUSION</b>Compounds 2-7 were isolated from S. cepharantha for the first time, and compound 7 was isolated from the genus Stephania for the first time, compound 4 was isolated from the Menispermaceae family for the first time.</p>

Diterpenoid alkaloids from roots of Aconitum recemulosum and their inhibitory effects on PAF-induced platelet aggregation / 中国中药杂志

<p><b>OBJECTIVE</b>To study diterpenoid alkaloids from the roots of Aconitum recemulosum, and their inhibitory effects on PAF-induced platelet aggregation.</p><p><b>METHOD</b>The root of A. recemulosum was extracted with 95% EtOH. The total alkaloids extracted were isolated and purified by several kinds of column chromatography over silica gel, RP-18, and Sephadex LH-20, and identified based on spectral analysis. And the inhibitory effects of isolated compounds on PAF-induced platelet aggregation were detected.</p><p><b>RESULT</b>Five alkaloids were isolated and identified as sachaconitine (1), 14-acetylsachaconitine (2), hemsleyanine C (3), circinasine A (4), and talatisamine (5). The results showed compounds 1 and 2 have moderate inhibition effect on PAF.</p><p><b>CONCLUSION</b>Compounds 1-5 were firstly isolated from this plant. Furthermore, compounds 1 and 2 possessed moderate inhibitory effects on PAF-induced platelet aggregation.</p>

Purification and Characterization of a Metalloproteinase with Weak Fibrinogenolytic Activity from Naja atra Venom / 中国生物化学与分子生物学报

A novel fibrinogenolytic protease,named atrase A,has been purified from the venom of Naja atra by sequential chromatography.Atrase A is a single chain glycoprotein with a molecular weight of 64.6 kD,an isoelectric point of pH 9.6 and a neutral sugar content of 4.16%.Atrase A specifically and slowly degraded α-chain of fibrinogen.This fibrinogenolytic activity Was inhibited by chelating agents(EDTA,EGTA and 1,10-phenanthroline)and DTY,and partially inhibited by PMSF,but not by soybean trypsin inhibitor,indicating it is a metalloproteinase.Atrase A showed edema-inducing activity and bactericidal activity against Staphylococcusa aureus.Atrase A did not show cytotoxicity on A549 and K562 cells in MTT assay,but detached adherent A549 cells from the substrate.Atrase A did not show significant inhibition of platelet aggregation induced by ADP or collagen,and did not exhibit proteolytic activities towards fibrin,azocasein and BAEE.It also did not show hemorrhage activity when injected subcutaneously into mice.

In vitro hemolyzation of a highly active anticomplement factor from the venom of Naja kaouthia / 中国药理学通报

AIM To study the hemolyzation of a highly active anti complementary protein (cobra venom factor, CVF) isolated from the venom of Naja kaouthia distributed in the south of Yunnan Province, China. METHODS Guinea pig red blood cell and serum were employed to evaluate the hemolyzation of this anticomplement factor, and the effects of temperature, pH, various bivalent mental ions and EDTA on its hemolyzation were investigated. RESULTS This anticomplement factor possessed hemolytic activity of 1 391 kU?g -1 protein. It showed high stability to heat, alkalinity and acidity. Ca 2+ , Mg 2+ , Mn 2+ promoted the hemolyzation of this anticomplement factor at 1, 2 and 5 mmol?L -1 . Zn 2+ , Co 2+ also promoted the hemolyzation of this anticomplement factor at 1 and 2 mmol?L -1 , but inhibited the hemolyzation at 5 mmol?L -1 . Cu 2+ strongly inhibited the hemolyzation at 1, 2 and 5 mmol?L -1 . EDTA also strongly inhibited the hemolyzation of this anticomplement factor. CONCL- USION The anticomplement factor showed hemolytic activity. This ability could be influenced by bivalent metal ions and EDTA, but rather stable when temperature or pH value changed.

Inhibitory effect of anticomplementary protein atrase B from cobra venom on platelet aggregation induced by activated complement / 中国药理学通报

Aim To investigate the inhibitory effect of atrase B on human platelet aggregation induced by activated complement.Methods By employing CVF to activate complement,the effect of atrase B on gel filtered platelet aggregation induced by activated complement was measured by turbidimetry and the expression of P-selectin and GPⅡb/Ⅲa on platelet membrane were detected by flow cytometry.Results Atrase B inhibited platelet aggregation and the expression of P-selectin and GPⅡb/Ⅲa on platelet membrane induced by activated complement.Conclusion Anticomplementary protein atrase B from Naja atra venom can significantly inhibit platelet activation and aggregation induced by activated complement.

Inflammatory mediators releasing and apoptosis of endothelial cell induced by cobra venom metalloproteinase atrase A / 中国药理学通报

Aim To investigate the effect of cobra venom metalloproteinase atrase A on human endothelial cell.Methods The effects of atrase A on the growth of HMEC were measured by MTT,SRB and morphological observation methods,respectively.After exposure to atrase A,IL-8,ICAM-1,MCP-1 and E-selectin released by HMEC were detected.Caspase-8 and caspase-3/7 were detected by fluorescent luminescence method.After intravenous injected atrase A to rats,the concentrations of ICAM-1,IL-8 and TNF-? in serum were measured.Results The results showed that atrase A(400,40 mg?L-1) significantly inhibited the growth of HMEC in low cell density.The microscopic examination revealed that atrase A detached the adherent HMEC.After exposure to atrase A,IL-8,ICAM-1 and MCP-1 released by HMEC were increased.Atrase A induced HMEC to express caspase-3/7 and caspase-8.After the administration of atrase A to rats,the concentrations of ICAM-1,IL-8 and TNF-? in serum were increased.There was no difference between the low-dose group and control group in our experiments.Conclusion Cobra venom metalloproteinase atrase A inhibits the growth of HMEC,and induces HMEC releasing inflammation mediators and apoptosis.

Study on microassay for screening ?-glucosidase inhibitors by orthogonal matrix method / 中国药理学通报

Aim To provide practical microassay for screening ?-glucosidase inhibitors in drug discovery.Methods The optimal conditions of assaying the activity of ?-glucosidase were determined in 96-well plates under physiological pH value and temperature by orthogonal matrix method.Reaction time and the concentrations of ?-glucosidase and substrate were optimized.After the effects of sample solvent(DMSO) used in the assay and stopping reagent on enzyme activity were assessed,the assay conditions were finally selected.Then 492 kinds of extracts from Guizhou ethno-drugs were screened.Results Practical and sensitive microassay for screening ?-glucosidase inhibitors was successfully constituted.And in 492 kinds of extracts,145 kinds of samples effectively inhibited the enzyme activity.Conclusion The microassay constituted in this work possesses advantages of being rapid,sensitive,reliable,cost saving,easy and flexible.

Comparative study on microassays for screening acetylcholinesterase inhibitors / 中国药理学通报

Aim To provide practical microassays for screening acetylcholinesterase (AChE) inhibitors in drug discovery.Methods The optimal conditions of assaying the activity of AChEs from electric eel,rat brain homogenate and cobra venom were determined in 96-well plates under physiological pH value and temperature by orthogonal matrix method.The concentrations of AChE,substrate and DTNB,and reaction time were optimized.After the effects of sample solvent (DMSO) used in the assay and stopping reagent on enzyme activity were assessed,the assay conditions were finally selected,and 492 kinds of extracts from Guizhou ethno-drugs were screened.Results Practical microassays for screening AChE inhibitors were successfully constituted by using AChEs mentioned above.The data analysis of screening results revealed that electric eel AChE possessed a high sentivity to inhibitors,and cobra venom AChE shared high similarity with rat brain homogenate in positive results.Conclusion Microassays constituted in this work possessed advantages of being easy,rapid,reliable,cost saving and flexible.AChE from electric eel was especially suitable for screening AChE inhibitors from extracts,and AChE from cobra venom was more suitable to be used in screening AChE inhibitors from large numbers of compounds.

Inhibitory effect and mechanism of cobra venom metalloproteinase atrase A on platelet aggregation / 中国药理学通报

Aim To investigate the effect and mechanism of cobra venom metalloproteinase atrase A on inhibiting platelet aggregation.Methods Platelet aggregation induced by collagen,ADP,PAF,AA,ristocetin and thrombin,respectively,was measured turbimetrically after platelet incubated with atrase A.Western blot was used to detect the effect of atrase A on cleavage of platelet membrane glycoprotein and von Willebrand Factor.Results Atrase A significantly inhibited platelet aggregation induced by ristocetin and thrombin in a dose-and time-dependent manner.Meanwhile,atrase A just showed slight inhibitory effect on platelet aggregation induced by PAF,AA,collagen,and ADP after incubated with PRP for 5 min.After incubation time was prolonged to 30 min,significant inhibition was shown on platelet aggregation.Western blot revealed that atrase A cleaved platelet membrane glycoprotein GPIb.Conclusions Cobra venom metalloproteinase atrase A significantly inhibits platelet aggregation induced by ristocetin and thrombin due to the cleavage of platelet membrane glycoprotein Ib.And atrase A also has inhibitory effect on platelet aggregation induced by ADP,PAF,AA,and collagen.

Study on microassay for screening butyrylcholinesterase inhibitors / 中国药理学通报

Aim To provide practical microassay for screening butyrylcholinesterase(BChE) inhibitors in drug discovery.Methods The enzyme reaction was optimized in 96-well plates under physiological pH value and temperature by orthogonal matrix method.After the assay conditions were finally selected,3 inhibitors and 115 extracts from Guizhou ethno-drugs were tested.Results A practical microassay for screening BChE inhibitors was successfully constituted by using rat serum as the source of BChE.Conclusion The microassay constituted in this work possess advantages of being practical,rapid,reliable and economical.

Pathogenesis of delayed xenograft rejection in pig-to-rhesus monkey cardiac transplantations / 中国免疫学杂志

Objective:To investigate the pahtogengesis and pathology of delayed xenograft rejection (DXR) after pig to rhesus monkey heart xenotransplantation.Methods:Heterotopic xenogeneic heart transplantation in the abdominal cavity was performed using piglet as donors.4 monkeys were used as recipients.Complete complement depletion was achieve in the recipients treated with repetitive doses of a high activity cobra venom factor (Y CVF).The recipients were immunosuppressed with a combination of cyclosporine A,cyclophosphamide and steroids.Sera were analyzed for C3,C4 levels and complement activity and anti pig endotheliocyte xenoantibody.The graft were examined histopathologically and immunohistochemically for C3,C4,C5b 9,IgM,IgG,necrosis factor alpha (TNF alpha),intercellular adhesion molecule 1 (ICAM 1),CD57 (NK cells),CD68 (macrophages),CD4 and CD8.Results:The xenografts survived 8,10,13,13 days respectively and all grafts occoured DXR.Venular thrombosis was outstanding feature within DXR xenografts,complicated with interstitial edma,local hemorrhage and myocardial necrosis,with mild to moderate cellular infiltration.The serum C3 levels and complement activity almost decreased to 0 from the day of transplantation due to Y CVF,the C4 level began to decrease 2 4 days before the cardic xenografts losing their function.The anti pig endotheliocyte xenoantibody also decreased after transplantation,and slightly increased during DXR,all rejected xenografts showed C3,C4,C5b 9,IgG and IgM deposits in different degree.Large numbers of macrophages (50% of total leukocytes) were found infiltrating the entire xenograft,a few natural killer cells (8%～10%),some of CD4+T cells (15%) and CD8+T cells (25%) were detcted also,up regulation of ICAM 1 on the graft endothelial cells and TNF alpha in the interstitial were demonstrated in the rejected heart.Conclusion:Both Humor and cell mediated immunologic reaction may play an important role in pahtogengesis of DXR. [