Hepatic ischemia/reperfusion injury can be modulated with thymoglobulin induction therapy

BACKGROUND: Thymoglobulin induction therapy has been shown to ameliorate delayed graft function and possibly decrease ischemia reperfusion injury in cadaver renal transplant recipients. This controlled randomized trial was designed to assess whether thymoglobulin also protects liver transplant recipients from ischemia reperfusion injury. PATIENTS AND METHODS: Twenty-two cadaver liver transplant recipients were randomized to receive either thymoglobulin (1.5 mg/kg per dose) during the anhepatic period and two doses every other day or no thymoglobulin. No differences in recipient or donor demographics were present. Maintenance immunosupression consisted of tacrolimus (or cyclosporine) and steroids for both groups. Donor biopsies were obtained during organ procurement, cold storage, and 1 hour after revascularization. Postoperative liver function tests were monitored. Early graft function, length of stay, patient and graft survival rates, incidence of primary nonfunction, and rate of rejection were assessed. RESULTS: Patient and graft survival at 3 months was 100%. There was no incidence of primary graft nonfunction and no need for retransplantation. The incidence of acute rejection was similar between the two groups. Although donor livers randomized to thymoglobulin had less optimal preimplantation biopsies, these recipients had significant decreases in ALT at day 1 compared to the control group (P = .02), near significant decreases of total bilirubin at day 5, and shorter length of hospitalization. CONCLUSION: Thymoglobulin allowed for more compromised liver grafts to be transplanted with less clinical evidence of ischemia reperfusion injury and improved function.     

Sulfatide and monoclonal antibodies prevent reperfusion injury in skin flaps

Sulfatide binds to P- and L-selectin, which play important roles in the initiation of neutrophil-endothelial interactions. Sulfatide protects skin flaps from ischemia-reperfusion injury. The purpose of this study was to evaluate the augmented protection when anti-rat ICAM-1 and anti-rat LFA-1 antibodies are combined with sulfatide in the ischemia-reperfusion model of rat skin flaps. Sulfatide was administered intravenously just before elevation of the right abdominal epigastric flap, and monoclonal antibodies were injected 30 min before clamp release. The femoral artery and vein were clamped above and below the epigastric vessels for 11 h and then the clamp was released. The administration of both sulfatide and monoclonal antibodies significantly increased the flap surviving area (6.58 +/- 0.61 cm(2) versus the group with monoclonal antibodies alone, 4.43 +/- 0.32 cm(2), P = 0.01). In the untreated rats the area was 1.86 +/- 0.36 cm(2). Histological examination 24 h after reperfusion in the group treated with sulfatide and monoclonal antibodies showed only slight leukocyte invasion into the flap, and myeloperoxidase activity 24 h after reperfusion was significantly reduced. This study indicates that both sulfatide and monoclonal antibodies protect rat skin flaps from ischemia-reperfusion injury.     

抗肿瘤坏死因子-α单克隆抗体减轻骨骼肌缺血再灌注损伤的研究

目的　研究抗肿瘤坏死因子 α(TNF α)单克隆抗体对骨骼肌缺血再灌注损伤的作用。方法　SD大鼠 2 4只 ,建立后肢缺血再灌注模型 ,抗TNF α单克隆抗体用量为 2mg/kg体重。采用酶联免疫吸附试验 (ELISA )法、比色法、免疫组织化学、流式细胞计数及电镜观察分别测定血浆TNF α、组织过氧化物酶 (MPO)、血管内皮细胞粘附分子 (ICAM ) 1、中性粒细胞CD18表达及超微结构改变。结果　应用抗TNF α单克隆抗体后血浆TNF α水平显著降低 (P <0 .0 1)。组织MPO(骨骼肌 :2 .11± 0 .2 5vs 4.2 8± 0 .5 5 ,P <0 .0 1;肺 :0 .93± 0 .0 1vs 2 .62± 0 .10 ,P <0 .0 1)、血管内皮细胞ICAM 1及中性粒细胞CD18(4 8.75± 9.2 8vs 1.13± 0 .2 0 ,P <0 .0 1)均明显下降 ,组织结构损伤减轻。结论　抗TNF α单克隆抗体能减轻骨骼肌缺血再灌注损伤。     

Platelet factor 4 release in patients undergoing cardiopulmonary resuscitation - can reperfusion be impaired by platelet activation?

Background: Reperfusion following cardiac arrest is associated with a marked activation of blood coagulation. This seems to be associated with microcirculatory reperfusion disorders. The present study was designed to investigate the possible involvement of platelets in reperfusion injury following cardiac arrest. Plasma levels of platelet factor 4 (PF 4) were used as an indicator for in vivo platelet activation because PF 4 is known to be released from platelets during aggregation.
Methods: Plasma PF 4 levels (normal range: < 5 IU/mL) were measured in 18 patients at predetermined time points during cardiopulmonary resuscitation (CPR). In the case of restoration of spontaneous circulation, additional blood samples were analyzed until seven days after stabilization. The PF 4 levels of four sex-matched volunteers were used as controls.
Results: The median of the maximum individual PF 4 levels measured during CPR was 27.5 IU/mL (range 1.2 to 90 IU/mL; P < 0.01 versus controls). Compared with PF 4 levels in control volunteers (median: 0.35 IU/mL; range 0.2 to 0.6 IU/mL), PF 4 levels were significantly elevated in patients during CPR and in the early phase until 24 hours after restoration of spontaneous circulation ( P < 0.05).
Conclusion: A marked increase in PF 4 levels was observed during CPR and in the early phase after cardiac arrest in man. This increase in PF 4 levels has to be viewed as an indicator of platelet activation, which may play a role in the etiology of reperfusion injury and microcirculatory reperfusion disorders occurring after cardiac arrest.     

Primary renal lymphoma in a patient with IgM monoclonal gammapathy

Reports on primary renal lymphoma are scarce in the urological literature, the most part of them are secondary on a lymphomatous infiltration of the kidneys. We report the case of a 77 year old man with an incidental mass on the kidney. After radiological studies (CT), we practise nephrectomy with a pathological result of a non-Hodgking B primary lymphoma. The patient present a IgM monoclonal gammapathy who need complementary treatment with chemotherapy. A literature review on currently recommended diagnostic and treatment practices in presented.     

Central venous catheter failure is induced by injury and can be prevented by stabilizing the catheter tip

Purpose: Thrombosis associated with central venous catheters is a significant cause of device failure, morbidity, and loss of access sites. We hypothesized that central vein thrombosis is caused by catheter injury to the vein wall and that it can be reduced by stabilizing the catheter tip. To test these hypotheses, we studied central vein catheters in a porcine model. Test catheters had a silicone-encased stainless steel loop at the indwelling end that contacted the vein wall and stabilized the catheter tip in the center of the vessel. Methods: Sealed silicon elastic (Silastic) catheters (3.2 mm outer diameter) with and without a stabilizing loop were inserted via the external jugular vein into the superior vena cava just above the right atrium. Animals were killed at 1, 2, 4, and 8 weeks, and the vena cava was inspected for the presence of thrombus and entrapment of the catheter tip. Results: In control animals mural thrombus developed at the site of the catheter tip. This thrombus organized by invasion of macrophages and smooth muscle cells, eventually forming a lesion similar to intimal hyperplasia. Lesion cross-sectional area was significantly smaller in animals with loop catheters than in control animals at 2 weeks (1.2 ± 1.3 vs 34.5 ± 23.9 mm2; p = 0.05) and 4 weeks (2.8 ± 0.3 vs 13.9 ± 5.8 mm2; p < 0.05). By 8 weeks the vena cava was nearly occluded in most animals and the catheter tip was entrapped in this lesion in all cases. Test catheters eliminated the injury process for up to 8 weeks (p < 0.01, χ2 control vs loop catheter entrapment). Very little injury response was found where the loop contacted the vein wall, and the catheter tip was free of thrombus in all cases. Conclusions: Mural thrombosis at the tip of indwelling central catheters is caused by chronic mechanical venous wall injury. Vessel injury and the resulting thrombosis can be prevented by a catheter modification that stabilizes the tip. Such a catheter may significantly reduce catheter malfunction and morbidity associated with these devices. (J Vasc Surg 1998;28:59-66.)     

Glucagon-like peptide 1 can directly protect the heart against ischemia/reperfusion injury

Glucagon-like peptide 1 (GLP-1), a gut incretin hormone that stimulates insulin secretion, also activates antiapoptotic signaling pathways such as phosphoinositide 3-kinase and mitogen-activated protein kinase in pancreatic and insulinoma cells. Since these kinases have been shown to protect against myocardial injury, we hypothesized that GLP-1 could directly protect the heart against such injury via these prosurvival signaling pathways. Both isolated perfused rat heart and whole animal models of ischemia/reperfusion were used, with infarct size measured as the end point of injury. In both studies, GLP-1 added before ischemia demonstrated a significant reduction in infarction compared with the valine pyrrolidide (an inhibitor of its breakdown) or saline groups. This protection was abolished in the in vitro hearts by the GLP-1 receptor antagonist exendin (9-39), the cAMP inhibitor Rp-cAMP, the PI3kinase inhibitor LY294002, and the p42/44 mitogen-activated protein kinase inhibitor UO126. Western blot analysis demonstrated the phosphorylation of the proapoptotic peptide BAD in the GLP-1-treated groups. We show for the first time that GLP-1 protects against myocardial infarction in the isolated and intact rat heart. This protection appears to involve activating multiple prosurvival kinases. This finding may represent a new therapeutic potential for this class of drug currently undergoing clinical trials in the treatment of type 2 diabetes.     

Human IgM xenoreactive natural antibodies can induce resistance of porcine endothelial cells to complement-mediated injury

Abstract: It is thought that human IgM xenoreactive natural antibodies (nAbs) can induce activation of porcine endothelial cells independent of complement. Therefore we hypothesized that pretreatment of porcine endothelial cells with anti-pig nAbs may affect the ability of the endothelial cells, when subsequently incubated with a source of nAbs and complement, to bind antibodies and complement components and to undergo complement-mediated cytotoxicity. We preincubated porcine endothelial cells at 37°C for 1 hr or 40 hr with a source of nAbs. We then incubated these pretreated endothelial cells with a complement source that contained a normal complement level and a low level of IgM nAbs for 1 hr to measure bound IgM and IgG and complement components, and for 4 hr to measure cytotoxicity. We found that preincubation for as long as 40 hr did not impair the binding of IgM and IgG, implying no antibody-induced loss of membrane antigens from the endothelial cells, or the binding of C3bi, C4d, C6, and C9 upon complement activation. In contrast, preincubation for 40 hr with a nAb source induced in the endothelial cells marked resistance to complement-mediated killing. Resistance could be induced with purified human IgM but not with purified IgG or IgM-depleted human serum. The ability of purified IgM to induce resistance was abrogated by removal of anti-pig xenoreactive nAbs by absorption with pig endothelial cells, and induction of resistance required protein synthesis. We conclude that prolonged incubation of human anti-pig nAbs with pig endothelial cells does not cause loss of endothelial cell membrane antigens or impairment in binding of nAbs or complement components; instead, it induces marked resistance to complement-mediated cytotoxicity. These observations may be of value to develop strategies that enhance survival of a xenograft.     

Preoperative bombesin administration can protect the rat small bowel allograft from ischemic reperfusion injury

Background/Purpose

Ischemic reperfusion injury (IR/I) should be minimum for the success of small bowel transplantation (SBTx). This study investigated whether preoperative administration of neuropeptide bombesin (BBS) had a protective effect against IR/I and subsequent acute rejection.

Methods

Allogenic SBTx was performed heterotopically in rats (n = 18). All rats were administered FK506 (0.32 mg/kg per day) everyday. The rats were divided into 3 groups of 6 rats each: group 1, BBS(−)5: warm ischemic time (WIT), 5 minutes without BBS; group 2, BBS(−)15: WIT, 15 minutes without BBS; group 3, BBS(+)15: WIT, 15 minutes with BBS. The specimens were obtained from the stoma site at 1 hour after reperfusion and on postoperative day (POD) 1 and 7. The graft mucosal state and degree of acute rejection were evaluated by H&E staining. The apoptotic cells in the crypt lesion was evaluated using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling immunohistochemistry. Apoptotic index (AI) was calculated for quantitative analysis.

Results

H&E staining revealed that the mucosal villi on POD 1 remained shortened in the BBS(−)15 group than in the other two groups. One hour after reperfusion, the AI in the BBS(−)15 group was 145.0‰ ± 37.2‰, which was significantly higher (P < .05) than in the BBS(−)5 group (32.6‰ ± 5.0‰) or the BBS(+)15 group (32.0‰ ± 3.0‰). On POD 7, the AI in the BBS(−)15 group was 63.7‰ ± 5.03‰, which was significantly higher (P < .05) than in the BBS(−)5 (17.3‰ ± 4.6‰) or the BBS(+)15 group (12.3‰ ± 3.06‰).

Conclusions

Even a short WIT of 15 minutes induced considerable allograft mucosal damage, which also worsened acute rejection. Exogenous BBS could prevent mucosal damage by IR/I and was also beneficial for the prevention of acute rejection.     

Attenuation of skeletal muscle reperfusion injury with intravenous 12 amino acid peptides that bind to pathogenic IgM

BACKGROUND: The injury sustained by reperfused skeletal muscle is inflammatory and is initiated by binding of pre-formed IgM to involved tissue, followed by local complement activation and further inflammation. A clone of natural IgM has been described that initiates this injury, suggesting that specific antigens are exposed on ischemic tissues that act as ligands for this pathogenic antibody. In these experiments, we examine the properties of short peptide sequences, and their homologues, that bind to the antigen-combining site of this pathogenic IgM clone. METHODS: A 12-mer phage display library was biopanned with the pathogenic IgM clone and then negatively selected against an inactive natural IgM clone. All 8 clones that bound specifically to the pathogenic IgM had closely related amino acid sequences. P8 is the clone that bound most avidly. Tissue lysates from ischemic tissue were reacted with pathogenic IgM, and immune complexes isolated and analyzed on SDS-PAGE. Bands were excised and sequenced, identifying non-muscle myosin as the protein reacting with pathogenic antibody in ischemic gut and glycogen phosphorylase as the counterpart in ischemic skeletal muscle. Both proteins contain sequence homologous to P8; N2 and GP1 are the natural 12-mers homologues that are contained within non-muscle myosin and glycogen phosphorylase, respectively. Wild-type C57/Bl6 mice, divided into groups receiving saline, P8, N2, GP1, or a random peptide at the start of the experiment, were subjected to 2 hours of tourniquet induced hind limb ischemia and 3 hours of reperfusion. Muscle was assessed for injury with histology and for immune activation with histochemistry. RESULTS: Intravenous administration of P8, N2, and GP1 led to significant attenuation of muscle injury (13 +/- 1.8 injured fibers/50 counted, 12 +/- 0.81, 8.0 +/- 0.73 respectively) after reperfusion injury compared to animals receiving saline (26 +/- 2.3) or the same mass of a random peptide (22 +/- 2.3), P less than .05. This level of protection from injury is comparable to that seen in the absence of antibody altogether. As well, P8-treated animals exhibited a marked decrease in deposition of IgM (as well as C3) in comparison to saline treated controls. CONCLUSIONS: Specific peptide blockade of an injury-inducing IgM clone decreased the local consequences of skeletal muscle ischemia/reperfusion injury in wild-type animals that have the full repertoire of IgM specificities. This indicates that the antibodies that initiate reperfusion injury have specificity only for P8-related antigens. This could also indicate that the variety of relevant ischemic antigens is quite restricted.     

Consistent immunostaining for cyclin D1 can be achieved on a routine basis using a newly available rabbit monoclonal antibody

Rabbit monoclonal antibody (MAb), which has become available only recently, theoretically combines the advantage of the high affinity attributable to its rabbit origin and the high specificity due to its monoclonal nature. Since immunohistochemical demonstration of cyclin D1 is notoriously difficult, this study aims to assess whether a newly available rabbit MAb against cyclin D1 (SP4) can improve the consistency of immunostaining, especially for the diagnosis of mantle cell lymphoma (MCL). A total of 150 cases of lymphoproliferative lesions, including 30 cases of MCL, histologic mimickers of MCL, and various types of lymphomas and leukemias, were studied. Immunostaining was performed on formalin-fixed, paraffin-embedded tissue sections using a labeled streptavidin-biotin peroxidase system in an automated immunostainer. All cases of MCL expressed cyclin D1, with a higher median staining score (8 out of a maximum of 12) compared with mouse MAb DCS-6 (score 4). In addition, 2 of 15 cases of B-cell chronic lymphocytic leukemia (B-CLL), 3 of 12 cases of multiple myeloma, and 2 of 5 cases of hairy cell leukemia were also positive. Comparable staining results could also be achieved by an optimized manual staining protocol. This study thus confirms the superior performance of the rabbit MAb SP4, which should permit consistent immunostaining for cyclin D1 to be readily achieved. The value of cyclin D1 immunohistochemistry in the differential diagnosis of MCL from other low-grade B-cell lymphomas is also affirmed, but with the caveat that rare cases of B-CLL can also be cyclin D1 positive.     

Epigallocatechin gallate can significantly decrease free oxygen radicals in the reperfusion injury in vivo

OBJECTIVE: There is experimental evidence that oxygen-derived free radicals (Superoxide (O(2)(-))) play a key role in tissue damage in ischemia-reperfusion injury. Among various antioxidants tested in vitro, natural polyphenols like Epigallocatechine gallate (EGCG) show a 164-fold higher scavenging activity for O(2)(-) than ascorbic acid We therefore conducted an animal study in order to investigate the impact of EGCG on O(2)(-) production during reperfusion after defined periods of ischemia in the muscle tissue of the rat, using a recently developed cytochrome c-based biosensor for on-line in vivo monitoring of O(2)(-). MATERIALS AND METHODS: Femoral artery and vein were dissected below the inguinal ligament in male Wistar rats. The cytochrome c-based biosensor was placed in the gastrocnemius muscle. Ischemia was induced by clamping the femoral vessels. Ischemia times were either 60 (n = 14) or 120 (n = 14) minutes. Six animals in each group received 4 mg/kg body weight EGCG intravenously at the time of reperfusion, another six animals in each group served as controls (no treatment). Additionally, two animals in each group received the same volume of saline instead of EGCG. The current response of the biosensor corresponding to the O(2)(-) concentrations in vivo was recorded on a PC. The gastrocnemius muscles were harvested for histological evaluation. RESULTS: The average maximum O(2)(-) concentration after 60 minutes of ischemia was 188, 18 nmol/L (23 pA) compared to 90 nmol/L (11 pA) (P <.01) with EGCG application. The mean O(2)(-) value after 120 minutes was 220 nmol/L (27 pA) versus 135 nmol/L (16.5 pA) (P <.01) with EGCG, respectively. Histological analysis showed advanced muscle cell injury and neutrophil infiltration in the group without EGCG. No O(2)(-) reduction could be verified administering saline instead of EGCG. CONCLUSION: For the first time the scavenging activity of an antioxidant was verified in vivo on-line. EGCG significantly diminished O(2)(-) tissue concentrations after 60 or 120 minutes of ischemia by an average of nearly 50%, suggesting its therapeutic potential.     

L-FABP can be an early marker of acute kidney injury in children

Background

Acute kidney injury (AKI) is a common postoperative complication following cardiopulmonary bypass (CPB) surgery. New biomarkers to identify patients with early AKI (before increases in serum creatinine) are needed to facilitate appropriate treatment. This study aimed to test the role of urinary liver fatty-acid-binding protein (L-FABP) as an early biomarker for AKI in children undergoing CPB surgery.

Methods

This is a case–control study of children undergoing CPB. AKI was defined as 50 % increase in serum creatinine at 48 h after surgery. For each patient, five serum and urine samples were obtained corresponding to time 0 h (presurgery) and 2, 6, 24, and 48 h after surgery.

Results

Twenty-seven patients, median age 360 days, were enrolled. AKI developed in 11 patients (41 %); three needed renal replacement therapy (peritoneal dialysis); there were two deaths. There were significant differences between patients with and without AKI in L-FABP levels at 2, 6, and 48 h after surgery, length of hospital stay, and CPB time; there were no differences in gender, patient age, and body weight. L-FABP was normalized to urinary creatinine concentration at all time points, with area under the receiver operator curve (AUC ROC) 0.867 at 2 and 6 h postoperatively. Correlation coefficient between L-FABP and length of hospital stay after surgery was statistically significant (r?=?0.722, p value?=?0.000).

Conclusions

Our results suggest that urinary L-FABP can be used to diagnose AKI earlier than rise in serum creatinine in children undergoing CPB.