[Value of four-dimensional echocardiography combined with speckle tracking technique on the assessment of right heart function and prognosis in patients with pulmonary arterial hypertension].

Objective: To investigate the value of four-dimensional echocardiography combined with speckle tracking technique on the assessment of right heart function and prognosis in patients with pulmonary arterial hypertension (PAH). Methods: In this prospective study, 51 patients with PAH diagnosed by right heart catheterization in east hospital and south hospital of Renji hospital affiliated to school of medicine of Shanghai Jiaotong University from September 2015 to July 2017 were enrolled as PAH group from July to November 2017. Meanwhile, 26 healthy volunteers with age and sex matched with pulmonary hypertension patients were recruited as control group. The patients were aged (45.8±15.5) years old in PAH group, and there were 6 males and 45 females. The healthy volunteers were aged (45.4±14.6) years old in control group, and there were 4 males and 22 females. Two-dimensional and four-dimensional echocardiographic images were obtained to measure the structure and function of the right heart. The myocardial strain of each ventricle and atrium was measured by sparkle tracking. The patients in PAH group were followed up from July 2017 to August 2018 to observe the endpoint events including all-cause death, re-hospitalization, and clinical deterioration. Results: There were significant differences in two-dimensional echocardiographic parameters(including tricuspid annular plane systolic excursion (TAPSE), right ventricular area change fraction (FAC), and right ventricular systolic pressure (RVSP)), speckle tracking parameters (including global longitudinal systolic peak strain of left ventricle (LVGLS), global longitudinal systolic peak strain of right ventricle (RVGLS),left atrial reservoir function(LASr), left atrial conduit function (LASc), left atrial pump function (LASp), right atrial reservoir function (RASr), right atrial conduit function (RASc), and right atrial pump function (RASp)), and four-dimensional echocardiographic parameters(including right ventricular end diastolic volume (RVEDV) , right ventricular end systolic volume(RVESV), right ventricular stroke volume(RVSV), right ventricular freewall longitudinal strain(RVLSf), interventricular septum longitudinal strain(IVSLS), right ventricular ejection fraction(RVEF)) between control group and PAH group (all P<0.01 or 0.05).Spearman correlation analysis showed that RVEF was correlated with 6-minute walking distance (r=0.540, P<0.001), B-type natriuretic peptide (r=-0.545,P<0.001), New York Heart Association (NYHA) cardiac function classification(r=-0.583, P<0.001), TAPSE(r=0.595, P<0.001), LVGLS (r=-0.461, P=0.001) ,LASc (r=0.453, P=0.002) ,RASc (r=0.532, P<0.001) ,RVESV (r=-0.418, P=0.004) , RVSV (r=0.351, P=0.017) , and IVSLS (r=-0.450, P=0.002) . Pearson correlation analysis also showed that RVEF was correlated with FAC(r=0.579, P<0.001),RVSP (r=-0.442, P=0.002) ,RVGLS (r=-0.521, P<0.001) , LASr (r=0.483, P=0.001) , RASr (r=0.617, P<0.001) , RASp (r=0.513, P<0.001) , and RVLSf (r=-0.592, P<0.001) .After a follow-up of (10.4±2.7) months, there were 4 all-cause deaths, 5 re-hospitalizations and 5 clinical deterioration. Multivariate Cox regression analysis showed that increased RVEF was independent protective factor for end-point events in PAH patients (HR=0.702, P=0.043), and increased RVSP was independent risk factor for end-point events in PAH patients (HR=1.083, P=0.017). The receiver operating characteristic (ROC) curve showed that RVEF and RVSP could be used to predict the end-point events in PAH patients. The area under the curve (AUC) was 0.835(P=0.001) and 0.820(P=0.001), respectively. Conclusions: RVEF measured by four-dimensional echocardiography is correlated with right ventricular function parameters measured by two-dimensional echocardiography and can be used to estimate the prognosis of PAH patients. The right atrial and left atrial function assessed by speckle tracking can also reflect the right ventricular function to a certain extent.

A Soluble Form of P Selectin Glycoprotein Ligand 1 Requires Signaling by Nuclear Factor Erythroid 2-Related Factor 2 to Protect Liver Transplant Endothelial Cells Against Ischemia-Reperfusion Injury.

Liver endothelial cell (LEC) damage is essential in the pathogenesis of ischemia-reperfusion injury (IRI) in transplant recipients. We analyzed the mechanism of LEC resistance against IRI by using a novel recombinant soluble form of P selectin glycoprotein ligand 1, tandem P selectin glycoprotein ligand immunoglobulin (TSGL-Ig), in a mouse model of hepatic cold preservation (4°C in University of Wisconsin solution for 20 h) and syngeneic orthotopic liver transplantation (OLT). Unlike controls, TSGL-Ig protected orthotopic liver transplants against ischemia-reperfusion (IR) stress, shown by depressed serum alanine aminotransferase levels, well-preserved hepatic architecture, and improved survival (42% vs. 92%). TSGL-Ig suppressed neutrophil/macrophage sequestration and proinflammatory cytokine/chemokine programs in OLT. Treatment with TSGL-Ig mitigated LEC activation (P and E selectin, VCAM-1 and intercellular adhesion molecule 1 expression). In parallel in vitro studies, TSGL-Ig diminished cellular damage in H2 O2 -stressed LEC cultures (lactic acid dehydrogenase and alanine aminotransferase levels). Increased thioredoxin, glutamate-cysteine ligase, NAD(P)H quinone dehydrogenase 1, and hypoxia-inducible factor 1α expression, along with transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), implied that TSGL-Ig exerts antioxidant functions in IR-stressed OLT and H2 O2 -stressed LECs. Indeed, Nrf2-deficient livers suffered fulminant IRI compared with WT despite concomitant TSGL-Ig therapy. Thus, TSGL-Ig is not only acting as a competitive antagonist blocking leukocyte migration into IR-stressed liver, but it may also act directly as an agonist stimulating Nrf2-mediated cytoprotection in LECs. This study supports the role of P selectin signaling in hepatic homeostasis in OLT, with broad implications for tissue damage conditions.

[Early detection of the cardiotoxicity induced by chemotherapy drug through two-dimensional speckle tracking echocardiography combined with high-sensitive cardiac troponin T].

Objective: To investigate the clinical value of two-dimensional speckle tracking echocardiography(2D-STE) combined with high-sensitive cardiac troponin T (hs-cTnT) in early detection of the cardiotoxicity induced by chemotherapy drug. Methods: Seventy-five non-Hodgkin's lymphoma patients who received the CHOP regimen were recruited in this study. Conventional echocardiography and 2D-STE were performed on these patients before chemotherapy, the second day after the third course of chemotherapy (during chemotherapy) and the second day after the last course of chemotherapy (after chemotherapy). The parameters included left ventricular ejection fraction (LVEF), global longitudinal strain (LS), global circumferential strain (CS) and global radial strain (RS). The serum hs-cTNT levels were tested simultaneously. Results: Three cycles of CHOP were completed in 30 patients and 6-8 cycles of CHOP were completed in 45 patients. The LVEF of 75 patients before, during and after chemotherapy was (63.8±2.6)%, (63.8±2.8)% and (64.0±3.3)%, respectively, without significant difference (P=0.91). However, the LS of 75 patients before, during and after chemotherapy was (-18.5±1.7)%, (-16.5±1.9)% and (-16.0±1.6)%, respectively. The CS was (-20.9±2.9)%, (-19.3±3.5)% and (-19.2±3.2)%, respectively. The RS was (39.2±6.4)%, (35.3±5.2)% and (35.0±6.2)%, respectively. The hs-cTnT was (0.001 0±0.002 0)ng/ml, (0.006 3±0.008 9)ng/ml and (0.007 3±0.003 8)ng/ml, respectively. The LS, CS and RS were significantly decreased while hs-cTnT was significantly increased during chemotherapy when compared to those before chemotherapy (all of P<0.01). Alternatively, the LS, CS, RS and hs-cTnT after chemotherapy were marginally different from those during chemotherapy (all of P>0.05). Moreover, T(LS-SD), T(CS-SD) and T(RS-SD) showed no significant difference before, during and after chemotherapy (all of P>0.05). The reduction of LS was positively associated with the enhancement of hs-cTnT after chemotherapy (r=0.60, P<0.01). Conclusion: 2D-STE combined with hs-cTnT can effectively and precisely detect the occult cardiotoxicity induced by anthracycline.

Fibronectin-α4ß1 interactions in hepatic cold ischemia and reperfusion injury: regulation of MMP-9 and MT1-MMP via the p38 MAPK pathway.

Liver ischemia-reperfusion injury (IRI) remains a challenging problem in clinical settings. The expression of fibronectin (FN) by endothelial cells is a prominent feature of the hepatic response to injury. Here we investigate the effects of the connecting segment-1 (CS-1) peptide therapy, which blocks FN-α4ß1 integrin leukocyte interactions, in a well-established model of 24-h cold liver IRI. CS-1 peptides significantly inhibited leukocyte recruitment and local release of proinflammatory mediators (COX-2, iNOS and TNF-α), ameliorating liver IRI and improving recipient survival rate. CS1 therapy inhibited the phosphorylation of p38 MAPK, a kinase linked to inflammatory processes. Moreover, in addition to downregulating the expression of matrix metalloproteinase-9 (MMP-9) in hepatic IRI, CS-1 peptide therapy depressed the expression of membrane type 1-matrix metalloproteinase (MT1-MMP/MMP-14) by macrophages, a membrane-tethered MMP important for focal matrix proteolysis. Inhibition of p38 MAPK activity, with its pharmacological antagonist SB203580, downregulated MMP-9 and MT1-MMP/MMP-14 expressions by FN-stimulated macrophages, suggesting that p38 MAPK kinase pathway controls FN-mediated inductions of MMP-9 and MT1-MMP/MMP-14. Hence, this study provides new insights on the role of FN in liver injury, which can potentially be applied to the development of new pharmacological strategies for the successful protection against hepatic IRI.

Disruption of Type-I IFN pathway ameliorates preservation damage in mouse orthotopic liver transplantation via HO-1 dependent mechanism.

Ischemia/reperfusion injury (IRI) remains unresolved problem in clinical organ transplantation. We analyzed the role of Type-I interferon (IFN) pathway in a clinically relevant murine model of extended hepatic cold preservation followed by orthotopic liver transplantation (OLT). Livers from Type-I IFN receptor (IFNAR) knockout (KO) or wild-type (WT) mice (C57/BL6) were harvested, preserved at 4°C in UW solution for 20 h and transplanted to groups of syngeneic IFNAR KO or WT recipients. Liver graft but not recipient IFNAR deficiency was required to consistently ameliorate IRI in OLTs. Indeed, disruption of Type-I IFN signaling decreased serum alanine aminotransferase (sALT) levels (p < 0.001), diminished Suzuki's score of histological OLT damage (p < 0.01) and improved 14-day survival (from 42%[5/12] in WT to 92%[11/12] in IFNAR KO; p < 0.05). Unlike in WT group, IFNAR deficiency attenuated OLT expression of TNF-α, IL-1ß, IL-6, MCP-1, CXCL-10, ICAM-1; diminished infiltration by macrophages/PMNs; and enhanced expression of antioxidant HO-1/Nrf2. The frequency of TUNEL+ apoptotic cells and caspase-3 activity/expression selectively decreased in IFNAR KO group. Small interfering (si)RNA-directed targeting of HO-1 restored cardinal features of liver IRI in otherwise resistant IFNAR-deficient OLTs. Thus, intact Type-I IFN signaling is required for hepatic IRI, whereas HO-1 is needed for cytoprotection against innate immunity-dominated organ preservation damage in IFNAR-deficient liver transplants.

[Analysis on infectivity of COVID-19 patients before and after last negative nucleic acid test].

Objective: To investigate the infection rate in close contacts of COVID-19 patients before and after the last negative nucleic acid test, evaluate the effect of dynamic nucleic acid test in determining the infectivity of COVID-19 patients. Methods: Dynamic nucleic acid test results of COVID-19 cases were collected in a retrospective cohort study. COVID-19 cases with negative nucleic acid test results before their first positive nucleic acid tests were selected as study subjects. Close contacts of the index cases and the secondary close contacts were kept isolation for medical observation to assess their risk of infection. Results: This study included 89 confirmed cases from two local COVID-19 epidemics in Ningbo. A total of 5 609 close contacts were surveyed, the overall infection rate was 0.20%. No close contacts of the COVID-19 cases before the last negative nucleic acid test were infected, and the infection rate in the close contacts of the COVID-19 cases after the last negative nucleic acid test was 1.33%, all of these close contacts lived together with the index cases. No secondary close contacts were infected. Conclusion: COVID-19 patient becomes infectious after the last nucleic acid is negative, and has no infectivity before the last nucleic acid negative.

[Preliminary analysis of the promotion effect of laparoscopic standardized surgical treatment for gastric cancer in regional medical centers in Shanghai].

Objective: To explore the promotion effect of laparoscopic standardized surgery for gastric cancer observational in some regional medical centers in Shanghai. Methods: A retrospective cohort study was carried out. Eleven regional medical centers in Shanghai received the promotion program of laparoscopic standardized surgery for gastric cancer, which was led by Ruijin Hospital, Shanghai Jiaotong University School of Medicine (Shanghai Minimally Invasive Surgery Center) from January to December 2020. Clinicopathological data of gastric cancer patients treated at these 11 regional medical centers before and after the promotion program were collected. Inclusion criteria were as follows: patients undergoing laparoscopic distal gastrectomy or total gastrectomy; gastric cancer confirmed by pathology; without distant metastasis or peritoneal metastasis. Patients who did not undergo laparoscopic D2 radical resection, or received neoadjuvant chemotherapy before surgery, or without complete clinical data were excluded. Patients undergoing laparoscopic surgery from January to December 2019 were included in the pre-promotion group (46 cases). Patients undergoing laparoscopic surgery from January to December 2021 were included in the post-promotion group (102 cases). In addition, patients undergoing laparoscopic surgery at Ruijin Hospital from January 2021 to December were included in the control group (138 cases). The baseline data, perioperative measurements postoperative complications, and pathological results of the three groups were analyzed and compared. Results: There were no significant differences in baseline characteristics among the three groups (all P>0.05). Compared with the pre-promotion group, the operation time in post-promotion group was significantly shorter [(207.3±36.0) minutes vs. (254.2±47.1) minutes, t=7.038,P<0.001], and the number of harvested lymph node was significantly more (24.4±12.2 vs. 18.9±5.5, t=2.900, P=0.004). However, there were no significant differences in the extent of resection, time to fluid intake, and postoperative hospital stay between the two groups (all P>0.05). Compared with the control group, the operation time [(207.3±36.0) minutes vs (172.6±26.0) minutes, t=8.281, P<0.001], time to fluid intake [(6.3±3.2) days than (5.5±3.0) days, t=2.029, P=0.044], and the postoperative hospital stay [(14.3±5.6) days vs. (10.1±4.8) days, t=6.036, P<0.001] in the post- promotion group were still longer. Total gastrectomy was less common in the post-promotion group compared with the control group [18 cases (17.6%) vs. 41 cases (29.7%), χ2=7.380, P=0.007]. However, there was no significant difference in the number of harvested lymph node between the two groups (P>0.05). The morbidity of postoperative complication in the post-promotion group (9.8%, 10/102) was significantly lower than that in the pre-promotion group (23.9%, 11/46) (χ2=5.183, P=0.023), while above morbidity was not significantly different between the post-promotion group and the control group [9.8% vs. 6.5% (9/138), χ2=0.867, P=0.352]. Conclusion: After the promotion of laparoscopic standardized surgery for gastric cancer in regional medical centers, the standardization degree of surgery has been improved, and the morbidity of postoperative complication decreases. Laparoscopic standardized surgery for gastric cancer can be promoted to more regional medical centers.

Sotrastaurin, a protein kinase C inhibitor, ameliorates ischemia and reperfusion injury in rat orthotopic liver transplantation.

Sotraustaurin (STN), a small molecule, targeted protein kinase C (PKC) inhibitor that prevents T-lymphocyte activation via a calcineurin-independent pathway, is currently being tested in Phase II renal and liver transplantation clinical trials. We have documented the key role of activated T cells in the inflammation cascade leading to liver ischemia/reperfusion injury (IRI). This study explores putative cytoprotective functions of STN in a clinically relevant rat model of hepatic cold ischemia followed by orthotopic liver transplantation (OLT). Livers from Sprague-Dawley rats were stored for 30 h at 4°C in UW solution, and then transplanted to syngeneic recipients. STN treatment of liver donors/recipients or recipients only prolonged OLT survival to >90% (vs. 40% in controls), decreased hepatocellular damage and improved histological features of IRI. STN treatment decreased activation of T cells, and diminished macrophage/neutrophil accumulation in OLTs. These beneficial effects were accompanied by diminished apoptosis, NF-κB/ERK signaling, depressed proapoptotic cleaved caspase-3, yet upregulated antiapoptotic Bcl-2/Bcl-xl and hepatic cell proliferation. In vitro, STN decreased PKCθ/IκBα activation and IL-2/IFN-γ production in ConA-stimulated spleen T cells, and diminished TNF-α/IL-1ß in macrophage-T cell cocultures. This study documents positive effects of STN on liver IRI in OLT rat model that may translate as an additional benefit of STN in clinical liver transplantation.

[Prognostic value of KIT and other clonal genetic mutations in core-binding factor acute myeloid leukemia].

Objective: To evaluate the prognostic significance of clonal gene mutations using next-generation sequencing in patients with core-binding factor acute myeloid leukemia (CBF-AML) who achieved first complete remission after induction chemotherapy. Methods: The study, which was conducted from July 2011 to August 2017 in First Affiliated Hospital of Soochow University, comprised 195 newly diagnosed patients with CBF-AML, including 190 patients who achieved first complete remission after induction chemotherapy. The cohort included 134 patients with RUNX1-RUNXIT1(+) AML and 56 patients with CBFß-MYH11(+) AML. The cohort age ranged from 15 to 64 years, with a median follow-up of 43.6 months. Overall survival (OS) and disease-free survival (DFS) were assessed by the log-rank test, and the Cox proportional hazards regression model was used to determine the effects of clinical factors and genetic mutations on prognosis. Results: The most common genetic mutations were in KIT (47.6% ) , followed by NRAS (20.0% ) , FLT3 (18.4% ) , ASXL2 (14.3% ) , KRAS (10.7% ) , and ASXL1 (9.7% ) . The most common mutations involved genes affecting tyrosine kinase signaling (76.4% ) , followed by chromatin modifiers (29.7% ) . Among the patients receiving intensive consolidation therapy, the OS tended to be better in patients with CBFß-MYH11(+) AML than in those with RUNX1-RUNXIT1 (+) AML (P=0.062) . Gene mutations related to chromatin modification, which were detected only in patients with RUNX1-RUNXIT1(+) AML, did not affect DFS (P=0.557) . The patients with mutations in genes regulating chromatin conformation who received allo-hematopoietic stem cell transplantation (allo-HSCT) achieved the best prognosis. Multivariate analysis identified KIT exon 17 mutations as an independent predictor of inferior DFS in patients with RUNX1-RUNXIT1(+) AML (P<0.001) , and allo-HSCT significantly prolonged DFS in these patients (P=0.010) . Conclusions: KIT exon 17 mutations might indicate poor prognosis in patients with RUNX1-RUNXIT1(+) AML. Allo-HSCT may improve prognosis in these patients, whereas allo-HSCT might also improve prognosis in patients with mutations in genes related to chromatin modifications.

Cytoprotective effects of a cyclic RGD peptide in steatotic liver cold ischemia and reperfusion injury.

The serious need for expanding the donor population has attracted attention to the use of steatotic donor livers in orthotopic liver transplantation (OLT). However, steatotic livers are highly susceptible to hepatic ischemia-reperfusion injury (IRI). Expression of fibronectin (FN) by endothelial cells is an important feature of hepatic response to injury. We report the effect of a cyclic RGD peptide with high affinity for the alpha5beta1, the FN integrin receptor, in a rat model of steatotic liver cold ischemia, followed by transplantation. RGD peptide therapy ameliorated steatotic IRI and improved the recipient survival rate. It significantly inhibited the recruitment of monocyte/macrophages and neutrophils, and depressed the expression of pro-inflammatory mediators, such as inducible nitric oxide synthase (iNOS) and interferon (IFN)-gamma. Moreover, it resulted in profound inhibition of metalloproteinase-9 (MMP-9) expression, a gelatinase implied in leukocyte migration in damaged livers. Finally, we show that RGD peptide therapy reduced the expression of the 17-kDa active caspase-3 and the number of apoptotic cells in steatotic OLTs. The observed protection against steatotic liver IRI by the cyclic RGD peptides with high affinity for the alpha5beta1 integrin suggests that this integrin is a potential therapeutic target to allow the successful utilization of marginal steatotic livers in transplantation.

Intestinal ischemia/reperfusion injury triggers activation of innate toll-like receptor 4 and adaptive chemokine programs.

BACKGROUND: Ischemia/reperfusion injury (IRI) is a major problem in intestinal transplantation. Toll-like receptor 4 (TLR4) has been implicated as a possible link between the innate and adaptive immune systems, however little data exists regarding TLR4 in intestinal IRI. The goal of this study is to evaluate the involvement of TLR4 in intestinal IRI and to assess the effect on T cell related chemokine programs. METHODS: C57BL6 mice underwent 100 minutes of warm intestinal ischemia by SMA clamping. Control WT mice underwent laparotomy without vascular occlusion. Separate survival and analysis groups were performed, and intestinal tissue was harvested at 1 hour, 2 hours, 4 hours, and 24 hours post-reperfusion. Analysis included histology, CD3 immunostaining, myeloperoxidase activity, Western blot, and PCR. RESULTS: Survival was significantly worse in the IRI group vs control (50% vs. 100%). IRI caused severe histopathological injury including mucosal erosions and villous congestion and hemorrhage. Myeloperoxidase activity increased in a time-dependent manner after IRI (2.71 0.25 at 1 hour, 2.92 0.25 at 2 hours, 4 0.16 at 4 hours, 5.1 0.25 at 24 hours vs 0.47 0.11 controls, P < .05). Protein expression of TLR4 followed by NF-kappaB was increased after IRI. Additionally, mRNA production of IP-10, MIP-2, MCP-1, and RANTES was increased at all time-points, as was mRNA for ICAM-1 and E-selectin. CONCLUSION: This study is the first to demonstrate increased expression of TLR4 and NF-kappaB after warm intestinal IRI. This detrimental cascade may be initiated by TLR4 via NF-kappaB signaling pathways, implicating TLR4 as a potential therapeutic target for the prevention of intestinal IRI.

Upregulation of heme oxygenase-1 protects genetically fat Zucker rat livers from ischemia/reperfusion injury.

We examined the effects of upregulation of heme oxygenase-1 (HO-1) in steatotic rat liver models of ex vivo cold ischemia/reperfusion (I/R) injury. In the model of ischemia/isolated perfusion, treatment of genetically obese Zucker rats with the HO-1 inducer cobalt protoporphyrin (CoPP) or with adenoviral HO-1 (Ad-HO-1) significantly improved portal venous blood flow, increased bile production, and decreased hepatocyte injury. Unlike in untreated rats or those pretreated with the HO-1 inhibitor zinc protoporphyrin (ZnPP), upregulation of HO-1 by Western blots correlated with amelioration of histologic features of I/R injury. Adjunctive infusion of ZnPP abrogated the beneficial effects of Ad-HO-1 gene transfer, documenting the direct involvement of HO-1 in protection against I/R injury. Following cold ischemia/isotransplantation, HO-1 overexpression extended animal survival from 40% in untreated controls to about 80% after CoPP or Ad-HO-1 therapy. This effect correlated with preserved hepatic architecture, improved liver function, and depressed infiltration by T cells and macrophages. Hence, CoPP- or gene therapy-induced HO-1 prevented I/R injury in steatotic rat livers. These findings provide the rationale for refined new treatments that should increase the supply of usable donor livers and ultimately improve the overall success of liver transplantation.

Fibronectin-alpha4beta1 integrin interactions modulate p42/44 MAPK phosphorylation in steatotic liver cold ischemia-reperfusion injury.

We investigated the effects of the connecting segment-1 (CS1) peptide, which blocks fibronectin (FN)-alpha4beta1 integrin interactions upon cell signaling, leukocyte migration, and secretion of proinflammatory cytokines, in a well-established steatotic rat liver model using ex vivo cold ischemia followed by isotransplantation. In this model, CS1 peptides were administered through the portal vein of steatotic Zucker rat livers prior and after cold ischemic storage. Lean Zucker recipients of fatty orthotopic liver transplantation (OLT) received an additional 3-day course of CS1 peptides post-OLT. CS1 peptide-treated steatotic OLTs harvested at 1, 3, and 7 days showed moderated levels of p42/44 mitogen-activated protein kinase (MAPK) phosphorylation, comparable to those observed in steatotic naive livers. In contrast, p42/44 MAPK phosphorylation was found up-regulated in 1- to 3-day damaged control OLTs. However, 7-day control OLTs were characterized by virtually lack of p42/44 MAPK phosphorylation. Lack of p42/44 MAPK phosphorylation in 7-day control OLTs was correlated with massive presence of leukocytes in the grafts and elevated levels of proinflammatory cytokines. CS1 peptide-treated OLTs at 7 days showed a profound decrease in T-cell (10 +/- 3 vs 56 +/- 20, P < .03) and monocyte/macrophage (+/++ vs +++) infiltration and significantly reduced levels of cytokine expression, such as IL-2 (approximately sixfold), and IFN-gamma (approximately three- to fourfold), as compared with controls.

Cyclic RGD peptides with high affinity for alpha5beta1 integrin protect genetically fat Zucker rat livers from cold ischemia/reperfusion injury.

We tested a hypothesis that interactions between fibronectin (FN), a key extracellular matrix component, and its integrin alpha5beta1 receptor are important in the development of ischemia/reperfusion (I/R) injury of steatotic liver transplants. We examined the effect of a cyclic RGD peptide (cRGD), with high affinity for alpha5beta1 integrin, in a well-established steatotic rat liver model of ex vivo cold ischemia followed by isotransplantation. In this model, cRGD peptides were administered through the portal vein of steatotic Zucker rat livers prior to and after cold ischemic storage. Lean Zucker recipients of fatty orthotopic liver transplants (OLTs) received an additional course of cRGD peptides 1 hour posttransplantation. cRGD peptide therapy significantly inhibited the recruitment of monocyte/macrophages, and repressed the expression of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. Moreover, it resulted in selective inhibition of inducible nitric oxide synthase (iNOS), and MMP-9 expression. Importantly, cRGD peptide therapy improved the function and histologic preservation of steatotic liver grafts, extending their 14-day survival in lean recipients from 50% in untreated to 100% in cRGD-treated OLTs. Thus, cRGD peptide-mediated blockade of FN-alpha5beta1 interaction protects against severe I/R injury otherwise experienced by steatotic OLTs.

Blockade of fibronectin-alpha4beta1 adhesive interactions down-regulates cyclooxygenase-2 inducible nitric oxide synthase and prolongs recipient survival in a 24-hour model of cold hepatic ischemia-reperfusion injury.

We investigated the effects of the connecting segment-1 (CS1) peptide, which blocks fibronectin (FN)-alpha4beta1 integrin interactions, upon recipient survival and extent of tissue injury in a well-established rat liver model of ex vivo 24-hour cold ischemia followed by isotransplantation. In this model, CS1 peptides were administered through the portal vein of rat livers prior to and after cold ischemic storage. In addition, recipients of orthotopic liver transplants (OLT) received a dose of CS1 peptides 1 hour post-OLT. CS1 peptide therapy significantly inhibited the intragraft recruitment of T lymphocytes and neutrophil activation/infiltration, and repressed important mediators of inflammation, such as cyclooxygenase-2, and inducible nitric oxide synthase expression. Importantly, CS1 peptide therapy improved function/histological preservation of liver grafts and extended their 14-day survival from 50% in control to 100% in CS1-treated OLTs. Thus, CS1 peptide-mediated blockade of FN-alpha4beta1 interaction protects against severe ischemia-reperfusion injury experienced otherwise by OLTs. These novel findings document the potential of targeting FN-alpha4beta1 in vivo interaction for improving OLT outcomes.

Allochimeric therapy induces unique regulatory T cells that mitigate chronic rejection.

Induction of tolerance to an allogeneic graft without the need for nonspecific immunosuppression is a major goal of transplantation therapy. We have shown that treatment with molecularly engineered, allochimeric [alpha(1h)(1/u)]-RT1.Aa class I MHC antigens bearing donor-type Wistar-Furth (WF, RT1.A(u)) or Lewis (LEW, RT1A(1)) amino acid substitutions for host-type ACI (RTI.A(a)) sequences in the alpha(1)-helical region induces donor-specific tolerance to cardiac allografts in rat recipients. The mechanisms involved in the establishment and maintenance of specific allograft tolerance are still not fully understood. It is now clear that acquisition of transplantation tolerance is an active, highly regulated, multistep process. According to the pool size model of allograft tolerance, the allograft outcome, rejection, or tolerance often depend on the balance between cytopathic and regulatory T cells (T-regs). This study examined mechanisms of chronic rejection (CR) development on a model of cardiac transplant tolerance after adoptive transfer of T-regs followed by allochimeric therapy. Generation of T-regs was demonstrated in vitro by MLR coculture and confirmed by adoptive transfer of T cells from primary recipients to secondary hosts. To confirm the true nature of regulatory cells, we performed a second transfer into tertiary recipients. Unlike T-regs from tolerant hosts, T cells from naive rats did not prolong graft survival. Histological evaluation of T-regs-transfected groups showed absence of visible CR. In contrast, T-regs generated in recipients after high-dose cyclosporine treatment failed to inhibit CR in transferred singeneic recipients. Allochimeric therapy triggers generation of unique regulatory lymphocytes that mitigate development of chronic rejection through regulation of anti-inflammatory mechanisms and down-regulation of alloantibody response.

Donor MHC class I peptides in conjunction with self-epitopes induce donor-specific tolerance in a dose-dependent manner but unable to abrogate chronic rejection.

Understanding specific tolerance mechanisms is a primary goal of transplantation science. We have previously shown that hosts treated with MHC class I protein have donor sequences in the alpha1-helix of the alpha1 domain on a background of self-epitopes, resulting in the development of donor-specific tolerance. However, the nature of class I alloantigenic determinants that regulate the alloimmune response remains unclear. The alpha1-helical sequence of RT1.A,1 which shares RT1.A(u) sequences, was substituted in the RT1.A(a) molecule to produce the composite [alpha1h(l/u)]-RT1.A(a) MHC class I allochimeric molecule. Immunodominant epitopes were identified within the hypervariable region of the alpha1 domain of RT1.A(a) (ACI), RT1.Al (Lewis, LEW), and RT1.A(u) (Wistar Furth [WF]). To clarify the mechanisms of tolerance development through presentation of donor-type immunogenic epitopes and cryptic self-epitopes we used synthetic peptides corresponding to donor immunogenic determinants with peptides derived from recipient self-sequences (RT1.A(a)--aa 10 to 49 P1 and 91 to 120 P3; and P2 RT1.A(l/u) 50 to 90). ACI recipients of LEW and WF cardiac allografts were injected through the portal vein (PV) at day 0 with four doses (2, 0.5, 0.25, and 0.125 mg/rat) of three peptide mixtures in conjunction with subtherapeutic CsA (10 mg/kg for 3 days). Allograft survival was strongly dose-dependent. Only low-dose regimens were consistent in tolerance induction, but such therapy did not abrogate development of chronic rejection (CR), unlike allochimeric therapy with soluble MHC class I protein. Different effects of protein or synthetic peptide therapies on development of CR suggest that development of specific tolerance is an active immunologic process and it depends on the form of allogeneic epitopes presented.

Identification of early tolerance regulator genes induced by allochimeric therapy using microarray-based genomewide scan.

We have demonstrated that peri- or postoperative delivery of allochimeric [a1h(u)]-RT1.A(a) class I major histocompatibility complex molecules with donor-type (RT1A(u)) immunogenic epitopes presented in recipient-type (RT1A(a)) sequences induced donor-specific tolerance in ACI (RT1a) recipients of WF (RT1u) heart allografts. A genomic scan during the early posttransplant period was performed to elucidate the underlying operative mechanisms. A rat genome study after transplantation was carefully designed using Affymetrix Rat Genome 230 2.0 Array. The allochimeric treatment group is 3-day cyclosporine (CsA)-treated ACI recipients that accepted Wistar Furth RT1u cardiac allografts with postoperative dosage of allochimeric molecules, while the control is 3-day CsA-treated ACI recipients of WF cardiac allografts. All the samples were harvested 5 days after heart transplant as the early stage of tolerance detection. Following array data normalization and modeling, we compared the above two treatment groups and identified a total of 250 tolerance regulator genes induced by allochimeric molecules only.

Adenovirus-mediated gene transfer in the transplant setting: early events after orthotopic transplantation of liver allografts expressing TGF-beta1.

We hypothesized that adenovirus mediated gene transfer of TGF-beta1 into liver grafts would enhanced local expression of this recombinant protein and down-regulate inflammatory and alloreactive immune response. A full length DNA encoding the murine TGF-beta1 was used to replaced the E1 region of adenovirus type 5 (AdmTGF-beta1). Expression and protein production of biologically active murine TGF-beta1 was tested in AdmTGF-beta1-transduced Hep G2 cells and TGF-beta-sensitive MV1 cells. In the transplant setting, the replication-defective vector was used to perfused cold preserved ACI liver allograft prior to transplantation into Lewis recipients. Control livers were similarly perfused with cold lactated Ringer's solution and were followed without immunosuppression. Animals were sacrificed at 1, 3, and 5 days after transplantation. Intragraft cytokine levels of TNFalpha, and IFNgamma were determined using ELISA and quantitative PCR. TGF-beta1 ELISA of culture supernatants from AdmTGF-beta1 transduced hepatocyte cell line Hep G2 excreted TGF-beta1 in quantities directly correlated with multiplicity of infection (MOI, vector:hepatic cell ratio). The biological activity of the excreted recombinant protein was confirmed by growth inhibition of MV1 TGF-beta-sensitive cells. Enhanced production of TGF-beta1 in transduced allografts was associated with decreased levels of TNFalpha and IFNgamma when compared with nonimmunosuppressed controls. Adenovirus-mediated gene transfer of murine TGF-beta1 into hepatic cells results in the expression of biologically active protein. Transduction of allografts with TGF-beta1 down-regulates TNFalpha and IFNgamma production early after orthotopic transplantation. Graft transduction with TGF-beta1 offers a novel approach to study the effects of single immune modulator on alloreactive immune response, T cell function, and cytokine cascade.