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.

Improvements in early behavior of rat kidney allografts after treatment of the brain-dead donor.

OBJECTIVE: To improve the quality of organs from brain-dead donors by assessing the influence of alternative strategies on the early behavior of kidneys after transplantation into unmodified hosts. SUMMARY BACKGROUND DATA: Kidneys transplanted from living donors perform consistently better than those from cadaver sources. The authors have recently shown that donor brain death produces inflammatory changes in peripheral organs within hours, amplifies coincident ischemia-reperfusion injury, and accelerates acute and chronic rejection. Normalization of the graft by donor hormone treatment has hitherto been unsuccessful. METHODS: A standardized rat model of brain death was used. Experimental groups included recipients of allogeneic grafts from living and brain-dead donors (F344-->LEW). Donors were treated immediately after induction of brain death either with intravenous steroids, which block inflammatory cytokine release, or a soluble P-selectin glycoprotein ligand (sPSGL), which blocks initial selectin-mediated cellular adhesion. Kidney grafts were examined serially up to 10 days by morphology, immmunohistology, and reverse transcriptase-polymerase chain reaction. RESULTS: Overall survival of ummodified recipients of kidneys from brain-dead donors was significantly reduced versus living donors. Animals with organs from brain-dead donors that had received steroids or sPSGL survived significantly longer than those from untreated brain-dead donors. The intensity of ischemia-reperfusion injury and of acute rejection was reduced. Cellular infiltration and transcription of mRNA of representative proinflammatory mediators were diminished. CONCLUSIONS: Treatment of organ donors at the time of brain death markedly improves organ quality after kidney transplantation, upgrading it to that from a living donor.

A novel iron chelator in combination with a P-selectin antagonist prevents ischemia/reperfusion injury in a rat liver model.

BACKGROUND: Hepatic ischemia/reperfusion (I/R) injury is associated with early and late graft failure after liver transplantation. A major mechanism is leukocyte adhesion to endothelium followed by release of reactive oxygen intermediates. We examined whether desferriexochelin 772SM (D-Exo), a lipid soluble iron chelator that prevents hydroxyl radical formation, can enhance the capacity of recombinant P-selectin glycoprotein ligand immunoglobulin (rPSGL-Ig), a glycoprotein that binds to P-selectin and inhibits neutrophil adhesion, to protect against I/R injury in an ex vivo rat liver model. METHODS: Rat livers were harvested and stored for 6 hr at 4 degrees C in University of Wisconsin solution and then perfused with oxygenated whole blood for 2 hr. Three groups were studied (n=6 rats/group): an untreated control group; a group that received 0.4 mg/kg rPSGL-Ig intraportally at the time of harvest; and a group that received 0.4 mg/kg rPSGL-Ig plus 1 micromol D-Exo intraportally both at the time of harvest and at the onset of reperfusion. Liver portal venous blood flow was assessed during perfusion, and at the end of each experiment, liver samples were collected for blinded histological evaluation and biochemical analyses. RESULTS: Livers treated with D-Exo + rPSGL-Ig had significantly higher blood flow than livers treated with rPSGL-1Ig alone (P<0.05), and both treatment groups had higher blood flow than controls (P<0.001). Production of carbonyl proteins, a protein oxidation product, was significantly reduced in the D-Exo + rPSGL-1Ig group (P<0.02 vs. controls), but not in the rPSGL-Ig alone group. Total reduced glutathione was significantly higher than controls in the D-Exo + rPSGL-Ig group (P<0.001 vs. controls), but not in the rPSGL-Ig alone group, indicating less oxidative stress in the D-Exo-treated group. Production of malondialdehyde, an index of lipid peroxidation, was significantly less than controls in both treatment groups (P<0.03). Histopathological findings paralleled these results with Banffs scores of 3.3+/-0.5, 1.8+/-0.4, and 1.3+/-0.5 in the control, rPSGL-Ig alone, and D-Exo plus rPSGL-Ig groups, resp. CONCLUSION: rPSGL-Ig provides partial protection against I/R injury to ex vivo rat livers; however, the addition of D-Exo substantially increases protection by reducing oxidative injury. These findings may have clinical relevance in preventing the consequences of I/R injury after liver transplantation.

Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P- and E-selectin bound to SLe(X) and PSGL-1.

P-, E- and L-selectin constitute a family of cell adhesion receptors that mediate the initial tethering and rolling of leukocytes on inflamed endothelium as a prelude to their firm attachment and extravasation into tissues. The selectins bind weakly to sialyl Lewisx (SLe(X))-like glycans, but with high-affinity to specific glycoprotein counterreceptors, including PSGL-1. Here, we report crystal structures of human P- and E-selectin constructs containing the lectin and EGF (LE) domains co-complexed with SLe(X). We also present the crystal structure of P-selectin LE co-complexed with the N-terminal domain of human PSGL-1 modified by both tyrosine sulfation and SLe(X). These structures reveal differences in how E- and P-selectin bind SLe(X) and the molecular basis of the high-affinity interaction between P-selectin and PSGL-1.

Exploitation of the continuum between early ischemia/reperfusion injury and host alloresponsiveness: indefinite kidney allograft survival by treatment with a soluble P-selectin ligand and low-dose cyclosporine in combination.

BACKGROUND: We have shown previously that sPSGL, a soluble glycoprotein ligand for P and E selectins, reduces the events associated with ischemia/reperfusion injury of the kidney. In the present study, we have attempted to modulate differentially early inflammatory influences and later host alloresponsiveness in an LBNF1-Lewis renal graft model by treatment with sPSGL in combination with a marginally effective dose of cyclosporine (CsA). METHODS: Four experimental groups were studied: group 1=control animals receiving vehicle only; group 2=sPSGL monotherapy alone; group 3=low-dose CsA; group 4=sPSGL plus low-dose CsA. Grafts were removed at 1, 3, 5, and 7 days (n=3/time point) and assessed by histology, immunohistology, and reverse transcriptase-polymerase chain reaction. Long-surviving grafts in recipients of groups 3 and 4 were followed functionally for more than 28 weeks. RESULTS: Graft function was prolonged indefinitely in recipients in group 4, all of which survived for more than 200 days. In contrast, survival of animals in groups 1 and 2 was not increased substantially, whereas only 4 of 17 animals in group 3 (23.5%) survived more than 24 days (P<0.01). Five days after engraftment, necrosis was relatively minimal in group 4 organs but pronounced in those of the other groups. By immunohistology, numbers of infiltrating CD4+ and CD8+ T cells and ED1+ macrophages were significantly diminished in group 4 allografts compared with those of the other groups. Serial assessment of chemokine and cytokine mRNA expression confirmed these findings. The long-term effects of CsA treatment alone were compared with those of sPSGL in combination with CsA. Proteinuria remained virtually absent in group 4 recipients. Morphologically, the few long-surviving grafts in group 3 showed signs of chronic rejection; those in group 4 remained relatively normal. CONCLUSIONS: Although treatment with sPSGL alone showed no apparent influence on the acutely rejecting transplants, at least by the parameters examined in this study, it produced indefinite survival of kidney grafts when used in combination with low-dose CsA. The data support the influence of early nonspecific injury on later immunological rejection.

Effects of explosive brain death on cytokine activation of peripheral organs in the rat.

BACKGROUND: The success rate of transplanted organs from brain-dead cadaver donors is consistently inferior to that of living sources. As cadaver and living unrelated donors are equally genetically disparate with a given recipient, the difference must lie within the donor himself and/or the effects of organ preservation and storage. We have hypothesized that irreversible central nervous system injury may up-regulate proinflammatory mediators and cell surface molecules in peripheral organs to be engrafted, making them more prone to host inflammatory and immunological responses. METHODS: Rats undergoing surgically induced acutely increased intracranial pressure (explosive brain death) were followed for 6 hr. Their peripheral tissues were examined by reverse transcriptase polymerase chain reaction and immunohistology, serum factors were assessed by enzyme-linked immunosorbent assay, and the influence of inflammatory molecules in the blood stream was determined by cross-circulation experiments with normal animals. RESULTS: mRNA expression of both lymphocyte- and macrophage-associated products increased dramatically in all tissues. Similar factors in serum were coincidentally increased; these were shown to be active in vivo by cross-circulation with normal animals. The organs of all control groups, including animals with important ischemic injury and with hemorrhagic shock, were negative. Up-regulation of MHC class I and II antigens and the co-stimulatory molecule B7 suggests increased immunogenicity of the peripheral organs. These changes could be inhibited by: (i) administration of a recombinant soluble P-selectin glycoprotein ligand-Ig, a P- and E-selectin antagonist; and (ii) a fusion protein, cytotoxic T lymphocyte antigen 4-Ig, which blocks B7-mediated T-cell co-stimulation. CONCLUSIONS: Activation of peripheral organs following explosive brain death may be caused by various interrelated events, including the effects of massive acute central injury, hypotension, and circulating factors. Almost complete suppression of these changes could be produced by biological agents. Such interventions, if reproducible in humans, could improve the quality of organs from "marginal" donors, broadening the criteria for donor acceptance.

Reduction of hepatic ischemia/reperfusion injury by a soluble P-selectin glycoprotein ligand-1.

OBJECTIVE: The authors' goal was to determine the effects of specific binding and blockade of P- and E-selectins by a soluble P-selectin glycoprotein ligand-1 (PSGL-1) in rat models of hepatic in vivo warm ischemia and ex vivo cold ischemia. The authors also sought to determine the effect of selectin blockade on isograft survival in a syngeneic rat orthotopic liver transplant model. SUMMARY BACKGROUND DATA: Ischemia/reperfusion (I/R) injury is a major factor in poor graft function after liver transplantation, which may profoundly influence early graft function and late changes. It is hypothesized that I/R injury leads to the upregulation of P-selectin, which is then rapidly translocated to endothelial cell surfaces within 5 minutes of reperfusion of the liver, initiating steps leading to tethering of polymorphonuclear neutrophil leukocytes to the vascular intima. Local production by leukocytes of interleukin-1, tumor necrosis factor-alpha, or both induces P-selectin expression on the endothelium and continues the cascade of events, which increases cell adherence and infiltration of the organ. METHODS: To examine directly the effects of selectins in a warm hepatic I/R injury model, 100 microg of PSGL-1 or saline was given through the portal vein at the time of total hepatic inflow occlusion. The effects of PSGL-1 in cold ischemia were assessed using an isolated perfused rat liver after 6 hours of 4 degrees C storage in University of Wisconsin (UW) solution, with or without the instillation of PSGL-1 before the storage. To evaluate the effect of selectin blockade on liver transplant survival, syngeneic orthotopic liver transplants were performed between inbred male Sprague-Dawley rats after 24 hours of cold ischemic storage in UW solution. A separate group of animals received two doses of 100 microg of PSGL-1 through the portal vein before storage and before reperfusion of the transplanted liver. Recipient survival was assessed at 7 days, and the Kaplan-Meier product limit estimate method was used for univariate calculations of time-dependent recipient survival events. RESULTS: In an in vivo warm rat liver ischemia model, perfusion with PSGL-1 afforded considerable protection from I/R injury, as demonstrated by decreased transaminase release, reduced histologic hepatocyte damage, and suppressed neutrophil infiltration, versus controls (p < 0.05). When cold stored livers were reperfused, PSGL-1 reduced the degree of hepatocyte transaminase release, reduced neutrophil infiltration, and decreased histologic hepatocyte damage (p < 0.05 vs. UW-only controls). On reperfusion, livers treated with PSGL-1 demonstrated increased portal vein blood flow and bile production (p < 0.05 vs. UW-only controls). In addition, 90% of the rats receiving liver isografts stored in UW solution supplemented with PSGL-1 survived 7 days versus 50% of those whose transplanted syngeneic livers had been stored in UW alone (p < 0.05). CONCLUSIONS: Selectins play an important role in I/R injury of the liver. Early modulation of the interaction between P-selectin and its ligand decreases hepatocyte injury, neutrophil adhesion, and subsequent migration in both warm and cold rat liver ischemia models. In addition, the use of PSGL-1 before ischemic storage and before transplantation prevents hepatic injury, as documented by a significant increase in liver isograft survival. These findings have important clinical ramifications: early inhibition of alloantigen-independent mechanisms during the I/R damage may influence both short- and long-term survival of liver allografts.

The cytokine-adhesion molecule cascade in ischemia/reperfusion injury of the rat kidney. Inhibition by a soluble P-selectin ligand.

Ischemia/reperfusion (I/R) injury associated with renal transplantation may influence both early graft function and late changes. The initial (

Prevention of late renal changes after initial ischemia/reperfusion injury by blocking early selectin binding.

BACKGROUND: Increasing clinical evidence suggests that delayed initial function secondary to ischemia/reperfusion injury alone, and particularly in combination with early episodes of acute rejection, reduces kidney allograft survival over time. METHODS: We investigated changes developing over the long term following a standardized ischemia/reperfusion insult in a Lewis rat model. The left kidney was isolated in a uninephrectomized host and cooled, and the pedicle was clamped for 45 min. Animals were followed for 48 weeks after initial renal injury. Organs were removed serially (4, 8, 16, 24, 32, 40, and 48 weeks) for immunohistology and reverse transcriptase polymerase chain reaction. RESULTS: Progressive proteinuria developed after 8 weeks. By immunohistology, CD4+ leukocytes and ED-1+ macrophages infiltrated the ischemic organs in parallel with up-regulation of major histocompatibility complex class II antigen expression. Because macrophages have been shown to be critical in chronic changes in other models, they were examined primarily in these studies. By reverse transcriptase polymerase chain reaction, macrophage-derived, fibrosis-inducing factors (transforming growth factor-beta, interleukin 6, and tumor necrosis factor-alpha) remained highly and constantly expressed throughout the follow-up period. The long-term influence of initial treatment with the soluble form of P-selectin glycoprotein ligand-1, a soluble ligand for P- and E-selectin, was then examined. All functional and structural changes remained at relative baseline, similar to uninephrectomized controls. CONCLUSIONS: These data suggest that blocking the initial selectin-mediated step after ischemia/reperfusion injury, which triggers significant early cellular and molecular events, also reduces later renal dysfunction and tissue damage over time. In part, the findings may be explained by the sparing of functioning nephron units, which if destroyed or compromised by the original insult, may contribute to long-term graft failure. This approach may be important clinically in the transplantation of kidneys from non-heart-beating or marginal donors or organs experiencing prolonged ischemic times.

A sulfated peptide segment at the amino terminus of PSGL-1 is critical for P-selectin binding.

P-selectin glycoprotein ligand 1 (PSGL-1) is a mucin-like glycoprotein expressed on the surface of myeloid cells and serves as the high affinity counterreceptor for P-selectin. The PSGL-1-P-selectin interaction is calcium dependent and requires presentation of sialyl-Lewisx (sLex)-type structures on the O-linked glycans of PSGL-1. We report here the identification of a non-carbohydrate component of the binding determinant that is critical for high affinity binding to P-selectin. Located within the first 19 amino acids, this anionic polypeptide segment contains at least one sulfated tyrosine residue. We propose that this sulfotyrosine-containing segment of PSGL-1, in conjunction with sLex presented on O-linked glycans, constitutes the high affinity P-selectin-binding site.

Structure and expression of cloned murine IFN-alpha genes.

The mouse has an interferon-alpha (MuIFN-alpha) gene family containing at least four, and likely more than ten members. A segment of mouse chromosomal DNA and cDNAs encoding murine alpha IFNs have been cloned, and the sequence of two MuIFN-alpha DNAs determined. No intron was found in the chromosomal gene. The two coding sequences produced biologically active IFN when expressed in monkey cells under the control of an SV40 promoter, and in E.coli under the control of the ampicillinase promoter. MuIFN-alpha 1 had no detectable activity on human cells, while MuIFN-alpha 2 was 20% as active on human as on mouse cells.

Statistical study of death episodes due to gastroenteritis in Jamaica, 1963-1967

The number of deaths caused by gastroenteritis rather than the notification of the illness is a more readily available index of the incidence of this disease in Jamaica. In order to examine the pattern of gastroenteritis with respect to its seasonal incidence data on a monthly or other cyclic basis. However, in the absence of such incidence figures for Jamaica, in this paper we have used the figures for deaths from gastroenteritis to determine the monthly incidence of disease in Jamaican children up to five years of age during the period 1963-1967(AU)

An analysis of communicable diseases in Jamaica (abstract only)

The latest period for which detailed and final figures on causes of mortality are available is the year 1956. At this time among the 10 principal causes of death there were still 5 communicable diseases - pneumonia, gastro-enteritis, respiratory tuberculosis, malaria and syphilis. Infective and parasitic diseases accounted for 12.7 percent of total deaths but this represented a reduction of more than 50 percent over the 10 year period prior to 1956 and the specific death rates for these diseases, excepting gastro-enteritis, are declining steadily. The majority of deaths from pneumonia, gastro-enteritis, malaria and syphilis occurs in the 0-4 years age groups and the respiratory tuberculosis death rates are now highest in the older age groups. Of the other reportable diseases large increases have been noted in the number of cases of infectious hepatitis reported (AU)