Stephen R. Spellman

Reperfusion injury after focal cerebral ischemia: The role of inflammation and the therapeutic horizon

Recent evidence indicates that thrombolysis may be an effective therapy for the treatment of acute ischemic stroke. However, the reperfusion of ischemic brain comes with a price. In clinical trials, patients treated with thrombolytic therapy have shown a 6% rate of intracerebral hemorrhage, which was balanced against a 30% improvement in functional outcome over controls. Destruction of the microvasculature and extension of the infarct area occur after cerebral reperfusion. We have reviewed the existing data indicating that an inflammatory response occurring after the reestablishment of circulation has a causative role in this reperfusion injury. The recruitment of neutrophils to the area of ischemia, the first step to inflammation, involves the coordinated appearance of multiple proteins. Intercellular adhesion molecule-1 and integrins are adhesion molecules that are up-regulated in endothelial cells and leukocytes. Tumor necrosis factor-alpha, interleukin-1, and platelet-activating factor also participate in leukocyte accumulation and subsequent activation. Therapies that interfere with the functions of these factors have shown promise in reducing reperfusion injury and infarct extension in the experimental setting. They may prove to be useful adjuncts to thrombolytic therapy in the treatment of acute ischemic stroke.

The graft-versus-leukemia effect using matched unrelated donors is not superior to HLA-identical siblings for hematopoietic stem cell transplantation.

Do some patients benefit from an unrelated donor (URD) transplant because of a stronger graft-versus-leukemia (GVL) effect? We analyzed 4099 patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML) undergoing a myeloablative allogeneic hematopoietic cell transplantation (HCT) from an URD (8/8 human leukocyte antigen [HLA]-matched, n=941) or HLA-identical sibling donor (n=3158) between 1995 and 2004 reported to the CIBMTR. In the Cox regression model, acute and chronic GVHD were added as time-dependent variables. In multivariate analysis, URD transplant recipients had a higher risk for transplantation-related mortality (TRM; relative risk [RR], 2.76; P< .001) and relapse (RR, 1.50; P< .002) in patients with AML, but not ALL or CML. Chronic GVHD was associated with a lower relapse risk in all diagnoses. Leukemia-free survival (LFS) was decreased in patients with AML without acute GVHD receiving a URD transplant (RR, 2.02; P< .001) but was comparable to those receiving HLA-identical sibling transplants in patients with ALL and CML. In patients without GVHD, multivariate analysis showed similar risk of relapse but decreased LFS for URD transplants for all 3 diagnoses. In conclusion, risk of relapse was the same (ALL, CML) or worse (AML) in URD transplant recipients compared with HLA-identical sibling transplant recipients, suggesting a similar GVL effect.

Neuroprotection by a Bile Acid in an Acute Stroke Model in the Rat

Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid, is a strong modulator of apoptosis in both hepatic and nonhepatic cells, and appears to function by inhibiting mitochondrial membrane perturbation. Excitotoxicity, metabolic compromise, and oxidative stress are major determinants of cell death after brain ischemia-reperfusion injury. However, some neurons undergo delayed cell death that is characteristic of apoptosis. Therefore, the authors examined whether TUDCA could reduce the injury associated with acute stroke in a well-characterized model of transient focal cerebral ischemia. Their model of middle cerebral artery occlusion resulted in marked cell death with prominent terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling (TUNEL) within the ischemic penumbra, mitochondrial swelling, and caspase activation. Tauroursodeoxycholic acid administered 1 hour after ischemia resulted in significantly increased bile acid levels in the brain, improved neurologic function, and an approximately 50% reduction in infarct size 2 and 7 days after reperfusion. In addition, TUDCA significantly reduced the number of TUNEL-positive brain cells, mitochondrial swelling, and partially inhibited caspase-3 processing and substrate cleavage. These findings suggest that the mechanism for in vivo neuroprotection by TUDCA is, in part, mediated by inhibition of mitochondrial perturbation and subsequent caspase activation leading to apoptotic cell death. Thus, TUDCA, a clinically safe molecule, may be useful in the treatment of stroke and possibly other apoptosis-associated acute and chronic injuries to the brain.

Chronic GVHD risk score: a Center for International Blood and Marrow Transplant Research analysis.

Several risk factors are associated with increased mortality in patients with chronic graft-versus-host disease (cGVHD), but there is considerable variability in the reported factors. Therefore, we evaluated patient, transplantation, and cGVHD characteristics to develop a risk score in 5343 patients with cGVHD. Ten variables were identified as being significant in multivariate analysis of overall survival and nonrelapse mortality (NRM): age, prior acute GVHD, time from transplantation to cGVHD, donor type, disease status at transplantation, GVHD prophylaxis, gender mismatch, serum bilirubin, Karnofsky score, and platelet count. These 10 variables were used to build a cGVHD risk score, and 6 risk groups (RGs) were identified. The 5-year NRM was 5% (1%-9%) in RG1, 20% (19%-23%) in RG2, 33% (29%-37%) in RG3, 43% (40%-46%) in RG4, 63% (53%-74%) in RG5, and 72% (59%-85%) in RG6. The 5-year overall survival was highest at 91% (95% confidence interval [CI]:85%-97%) in RG1, followed by 67% (65%-69%) in RG2, 51% (46%-55%) in RG3, 40% (37%-43%) in RG4, 21% (12%-30%) in RG5, and 4% (0%-9%) in RG6 (all P < .01). This analysis demonstrates the usefulness of data from a large registry to develop risk-score categories for major transplantation outcomes. Validation of this cGVHD risk score is needed in a different population to ensure its broad applicability.

Immunization with Dendritic Cells Pulsed with Tumor Extract Increases Survival of Mice Bearing Intracranial Gliomas

The purpose of this study is to investigate the efficacy of dendritic cell-mediated immunotherapy against intracranial gliomas. Cloned DC2.4 dendritic cells originating from C57BL/6 mice were pulsed with glioma GL261 cell extracts and administered i.p. to C57BL/6 mice with intracranial GL261 gliomas. The survival of mice with and without pulsed dendritic cells was monitored after intracranial implantation of the GL261 glioma cells. Fluorescence activated cell sorting (FACS) analysis showed that DC2.4 cells express high levels of MHC class I and class II molecules, costimulatory molecules B7-l and B7-2, and the cell adhesion molecule ICAM-l. Antigen-presenting capabilities in these dendritic cells were initially characterized in vitro by a mixed lymphocyte reaction, showing that Balb/c CD4+ and CD8+ T cells were able to generate allogeneic responses to DC2.4 cells. Tumor extract-pulsed DC2.4 dendritic cells were then used for the treatment of C57BL/6 mice with syngeneic GL261 gliomas. Animals with intracranial GL261 gliomas and vaccinated i.p. with pulsed DC2.4 dendritic cells exhibited significantly enhanced survival, relative to animals treated with saline or non-pulsed DC2.4 cells alone. In addition, cured animals showed an increased delayed-type hypersensitivity response to GL261 cells and survived when rechallenged with intracranial GL261 gliomas. In summary these results indicate that dendritic cells pulsed with tumor extract can enhance immune responses to tumor antigen and therefore represent a potential immunotherapeutic approach for treating patients with intracranial gliomas.

HLA-Identical Sibling Compared With 8/8 Matched and Mismatched Unrelated Donor Bone Marrow Transplant for Chronic Phase Chronic Myeloid Leukemia

PURPOSEnTransplantation of hematopoietic stem cells from an unrelated donor (URD) is an option for many patients who do not have an HLA-identical sibling donor (MSD). Current criteria for the selection of URDs include consideration for HLA alleles determined by high resolution typing methods, with preference for allele-matched donors. However, the utility and outcome associated with transplants from URDs compared with those from MSDs remains undefined.nnnPATIENTS AND METHODSnWe examined clinical outcome after patients received bone marrow transplants (BMTs) from MSDs; HLA-A, -B, -C, and DRB1 allele-matched URDs (8/8); and HLA-mismatched URDs in a homogeneous population of patients with chronic myeloid leukemia (CML) in first chronic phase (CP1) where a strong allogeneic effect and hence a lower risk of relapse is anticipated. Transplantation outcomes were compared between 1,052 URD and 3,514 MSD BMT recipients with CML in CP1.nnnRESULTSnFive-year overall survival and leukemia-free survival (LFS) after receipt of BMTs from 8/8 matched URDs were worse than those after receipt of BMTs from MSDs (5-year survival, 55% v 63%; RR, 1.35; 95% CI, 1.17 to 1.56; P < .001; LFS, 50% v 55%; RR, 1.21; 95% CI, 1.06 to 1.40; P = .006). Survival was progressively worse with greater degrees of mismatch. Similar and low risk of relapse were observed after receipt of transplant from either MSD or URD.nnnCONCLUSIONnIn this homogeneous cohort of good risk patients with CML in CP1, 5-year overall survival and LFS after receipt of transplant from 8/8 allele-matched donors were modestly though significantly worse than those after receipt of transplant from MSDs. Additive adverse effects of multilocus mismatching are not well tolerated and should be avoided if possible.

Interleukin-12-based immunotherapy against rat 9L glioma

OBJECTIVEnInterleukin-12 (IL-12) may be useful for immunotherapy against gliomas because it can reverse the glioma-induced suppression of T-cell proliferation and interferon-gamma production. We postulated that peripheral infusion of IL-12 along with irradiated tumor cells can lead to immunological rejection of 9L glioma.nnnMETHODSn9L gliosarcoma flank tumors were established in syngeneic Fischer 344 rats. Osmotic minipumps delivered IL-12 subcutaneously, and irradiated 9L cells were injected on Days 0, 3, 7, 14, and 21. Tumor volumes were measured by a blinded observer. For tumor rechallenge, animals initially cured of 9L flank tumors received either another implantation of flank tumor or a stereotactic injection of 10(6) 9L cells into the right striatum. Delayed-type hypersensitivity was measured after injecting 10(6) irradiated 9L tumor cells into the right pinnae.nnnRESULTSnTumor growth curves were significantly different between treated and control animals. Among the animals that received 1 ng per day of IL-12, 40% did not develop any measurable tumors at all. A combination of irradiated 9L cells and IL-12 was necessary for optimal effect. Cured animals rejected future flank tumors. All animals rechallenged with intraparenchymal brain tumors survived, whereas control animals all died by Day 22. Delayed-type hypersensitivity measurements showed a specific and long-lasting response against 9L cells.nnnCONCLUSIONnContinuous administration of the lymphokine IL-12, in the presence of irradiated tumor cells for antigen presentation, circumvents the need for gene transfection for generating tumor cell vaccines. We have demonstrated that the combination of IL-12 and irradiated tumor cells can lead to regression of 9L flank tumors and resistance to future flank and central nervous system tumor challenges.

Effects of combined granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2, and interleukin-12 based immunotherapy against intracranial glioma in the rat.

Cytokines play a major role in the regulation of the immune system. Granulocyte–macrophage colony-stimulating factor (GM-CSF) has been shown to be useful for immunotherapy against glioma because it can stimulate dendritic cells to present tumor antigen. Interleukin-2 (IL-2) is involved in T-cell expansion, and interleukin-12 (IL-12) drives the T-helper cell type I response. Previous studies have shown that each of these cytokines alone can induce the regression of tumor cells. In the present study we postulated that peripheral infusion of GM-CSF along with either IL-2 or IL-12 and irradiated tumor cells can lead to increased survival from 9L brain tumors. 9L gliosarcoma cells (106) were implanted in the brains of syngeneic Fischer 344 rats. Osmotic minipumps were utilized for subcutaneous, continuous delivery of GM-CSF, either alone or with IL-2 or IL-12. Irradiated 9L cells were injected subcutaneously at various time points during treatment. Delayed-type hypersensitivity (DTH) and immunohistological analysis were used to further characterize the anti-tumor response. Treatment with GM-CSF and irradiated tumor cells led to an increase in survival rate in rats with intracranial 9L tumors when compared to untreated animals. The addition of IL-2 or IL-12 to the GM-CSF/tumor cell therapy further increased the survival rate up to 90%. The anti-tumor response was associated with vigorous DTH against 9L cells and increased infiltration of CD4+ and CD8+ lymphocytes into the tumor. These results suggest that the combined infusion of GM-CSF and other cytokines may be effective adjuvants in treating brain tumors.

Optimal Timing of Hemodilution for Brain Protection in a Canine Model of Focal Cerebral Ischemia

BACKGROUND AND PURPOSEnHemodilution is known to ameliorate the effects of focal ischemia when used shortly after cerebral arterial occlusion; however, it remains to be proved whether hemodilution will be effective when used at more clinically relevant times, ie, with some delay between the onset of ischemia and initiation of therapy.nnnMETHODSnThirty-two dogs were selected for inclusion in this study. Cerebral infarction was induced by permanent occlusion of the middle cerebral and the azygos anterior cerebral arteries. The animals were allocated to 1 of 4 groups of eight animals each: arterial occlusion without hemodilution (group 1); hemodilution immediately after occlusion (group 2); hemodilution 3 hours after occlusion (group 3); and hemodilution 6 hours after occlusion (group 4). Isovolemic hemodilution to a hematocrit of 30% was performed. The animals were killed 6 days after induction of ischemia, and the infarct size was determined.nnnRESULTSnGroups 2 and 3 showed significant reduction of infarct size (P < .0001) when compared with group 1. The neurological grade of group 3 on postoperative days 4, 5, and 6 was significantly better than those of groups 1 and 4 (P < .01). Group 4 showed a significant increase in the incidence of hemorrhagic infarction when compared with groups 1 and 2 (P < .01).nnnCONCLUSIONSnThe current study indicates that hemodilution administered as much as 3 hours after ischemia is effective in reducing infarct size and improving neurological status. When administered 6 hours after ischemia, hemodilution is not helpful and may be harmful.

Neuronal Protection from Cerebral Ischemia by Synthetic Fibronectin Peptides to Leukocyte Adhesion Molecules

Leukocytes play an important role in the development of ischemia/reperfusion injury. Recent work in our laboratory has demonstrated that a mixture of synthetic fibronectin peptides to leukocyte adhesion molecules reduces ischemic brain damage after transient focal cerebral ischemia. The purpose of this study was to evaluate the efficacy of the individual peptides on leukocyte accumulation, infarct size, and neurological outcome in rats subjected to 1 h of cerebral ischemia and 48 h of reperfusion. Thirty-five animals were divided into five groups: transient ischemia without treatment (Group I), treatment with arginyl-glycyl-aspartic acid (RGD) peptide (Group II), connecting segment (CS)-1 peptide (Group III), fibronectin (FN)-C/H-V peptide (Group IV), and scrambled FN-C/H-V peptide (Group V). Groups III and IV showed a significant decrease in the degree of leukocyte infiltration in the lesion and in the infarct size (p < 0.05) when compared to Groups I, II, and V. The neurological grade of Groups III and IV was significantly better than in Groups I, II, and V at 48 h after reperfusion (p < 0.01). Thus, in addition to demonstrating the potential efficacy of synthetic peptides as therapeutic agents for ischemia-reperfusion, these results also offer new insights into the mechanisms of leukocyte arrest and recruitment in ischemia/reperfusion injury.