Novel HLA-DP region susceptibility loci associated with severe acute GvHD.

Despite HLA allele matching, significant acute GvHD remains a major barrier to successful unrelated donor BMT. We conducted a genome-wide association study (GWAS) to identify recipient and donor genes associated with the risk of acute GvHD. A case-control design (grade III-IV versus no acute GvHD) and pooled GWA approach was used to study European-American recipients with hematological malignancies who received myeloablative conditioning non-T-cell-depleted first transplantation from HLA-A, -B, -C, -DRB1, -DQB1 allele level (10/10) matched unrelated donors. DNA samples were divided into three pools and tested in triplicate using the Affymetrix Genome-wide SNP Array 6.0. We identified three novel susceptibility loci in the HLA-DP region of recipient genomes that were associated with III-IV acute GvHD (rs9277378, P=1.58E-09; rs9277542, P=1.548E-06 and rs9277341, P=7.718E-05). Of these three single nucleotide polymorphisms (SNPs), rs9277378 and rs9277542 are located in non-coding regions of the HLA-DPB1 gene and the two are in strong linkage disequilibrium with two other published SNPs associated with acute GvHD, rs2281389 and rs9277535. Eighteen other recipient SNPs and 3 donor SNPs with a high level of significance (8E-07 or lower) were found. Our report contributes to emerging data showing clinical significance of the HLA-DP region genetic markers beyond structural matching of DPB1 alleles.

Directionality of non-permissive HLA-DPB1 T-cell epitope group mismatches does not improve clinical risk stratification in 8/8 matched unrelated donor hematopoietic cell transplantation.

In 8/8 HLA-matched unrelated donor (UD) hematopoietic cell transplants (HCT), HLA-DPB1 mismatches between alleles from different T-cell epitope (TCE) groups (non-permissive mismatches) are associated with significantly higher risks of mortality compared with those between alleles from the same TCE group (permissive mismatches); however, the relevance of mismatch directionality, that is (host vs graft (uni-directional HvG), graft vs host (uni-directional GvH) or both (bi-directional) in the non-permissive setting is unknown. We show here significantly higher in vitro relative responses (RR) to bi-directional mismatches compared with uni-directional HvG or GvH mismatches in a total of 420 one-way mixed lymphocyte reactions between 10/10 matched pairs (RR 27.5 vs 7.5 vs 15.5, respectively, P<0.001). However, in 3281 8/8 matched UD HCT for leukemia or myelodysplastic syndrome, the hazards of transplant-related mortality (TRM) were similar for uni-directional HvG or GvH mismatches and bi-directional mismatches (hazard ratio (HR) 1.32, P=0.001 vs HR 1.28, P=0.005 and HR 1.34, P=0.046), compared with permissive mismatches. Similar results were observed for overall survival. No statistical differences between the uni- and the bi-directional non-permissive groups were detected in pairwise comparisons for any of the outcomes tested. We conclude that consideration of directionality does not improve risk stratification by non-permissive HLA-DPB1 TCE mismatches in UD searches.

GvHD after umbilical cord blood transplantation for acute leukemia: an analysis of risk factors and effect on outcomes.

Using the Center for International Blood and Marrow Transplant Research (CIBMTR) registry, we analyzed 1404 umbilical cord blood transplantation (UCBT) patients (single (<18 years)=810, double (⩾18 years)=594) with acute leukemia to define the incidence of acute GvHD (aGvHD) and chronic GvHD (cGvHD), analyze clinical risk factors and investigate outcomes. After single UCBT, 100-day incidence of grade II-IV aGvHD was 39% (95% confidence interval (CI), 36-43%), grade III-IV aGvHD was 18% (95% CI, 15-20%) and 1-year cGvHD was 27% (95% CI, 24-30%). After double UCBT, 100-day incidence of grade II-IV aGvHD was 45% (95% CI, 41-49%), grade III-IV aGvHD was 22% (95% CI, 19-26%) and 1-year cGvHD was 26% (95% CI, 22-29%). For single UCBT, multivariate analysis showed that absence of antithymocyte globulin (ATG) was associated with aGvHD, whereas prior aGvHD was associated with cGvHD. For double UCBT, absence of ATG and myeloablative conditioning were associated with aGvHD, whereas prior aGvHD predicted for cGvHD. Grade III-IV aGvHD led to worse survival, whereas cGvHD had no significant effect on disease-free or overall survival. GvHD is prevalent after UCBT with severe aGvHD leading to higher mortality. Future research in UCBT should prioritize prevention of GvHD.

YKL-40 in allogeneic hematopoietic cell transplantation after AML and myelodysplastic syndrome.

YKL-40, also called chitinase-3-like-1 protein, is an inflammatory biomarker that has been associated with disease severity in inflammatory and malignant diseases, including AML, multiple myeloma and lymphomas. The objective of the current study was to assess the prognostic value of pretransplant recipient and donor plasma YKL-40 concentrations in patients with AML (n=624) or myelodysplastic syndrome (n=157) treated with allogeneic hematopoietic cell transplantation (HCT). In recipients, the plasma YKL-40 concentrations were increased when the HCT-comorbidity index was ⩾5 (P=0.028). There were no significant associations between plasma YKL-40 concentrations in recipients and any outcome measures. In donors with YKL-40 plasma concentrations above the age-adjusted 95th percentile, a trend toward increased grade II-IV acute GvHD in recipients was observed (adjusted hazard ratio 1.39 (95% confidence interval 1.00-1.94), P=0.050), with no significant associations with overall survival, treatment-related mortality or relapse. In conclusion, our study shows that YKL-40 does not aid risk stratification of patients undergoing allogeneic HCT, but suggests that YKL-40 may aid donor selection when multiple, otherwise equal, donors are available.

Laminin peptide ameliorates brain injury by inhibiting leukocyte accumulation in a rat model of transient focal cerebral ischemia.

Postischemic cerebral inflammation has been reported to contribute to ischemic brain damage. During inflammation, constituents of the extracellular matrix such as fibronectin and laminin are recognized by certain integrins or proteoglycans and play an important role in the cell adhesion process. The purpose of this study was to evaluate the efficacy of peptides derived from laminin on leukocyte accumulation, infarct size, and neurological outcome in rats subjected to 1 h of cerebral ischemia and 48 h of reperfusion. Forty-four animals were included in this study: transient ischemia without treatment (Group I), treatment with TG-1 peptide (Group II), GD-1 peptide (Group III), and GD-6 peptide (Group IV). Group II showed a significant reduction of the leukocyte accumulation (p < 0.001) and infarct size (p = 0.015) when compared with Group I. The neurological grade of Group II was also significantly better than in Group I at 48 h after reperfusion (p = 0.012). Based on these data, which are the first to explore the therapeutic potential of this peptide in cerebral ischemia, laminin peptide may offer a novel therapeutic approach to allaying injury in ischemic stroke.

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.

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.

Interleukin-12-based immunotherapy against rat 9L glioma.

OBJECTIVE: Interleukin-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. METHODS: 9L 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. RESULTS: Tumor 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. CONCLUSION: Continuous 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.

Antagonism of leukocyte adherence by synthetic fibronectin peptide V in a rat model of transient focal cerebral ischemia.

OBJECTIVE: Activated polymorphonuclear leukocytes (PMNs) seem to be directly involved in potentiating ischemic brain injury. Recent work in our laboratory demonstrated that synthetic fibronectin peptides significantly inhibit PMN accumulation in ischemic tissue, reduce the size of infarction, and reduce neurological dysfunction after transient focal cerebral ischemia in rats. The purpose of this study was to examine any dose-related effects (Experiment 1) and the optimal timing of the administration (Experiment 2) of synthetic fibronectin peptide V (FN-C/H-V) to further substantiate the role of the peptide in ameliorating cerebral ischemic damage. METHODS: Fifty-six animals were included in the study. We evaluated the efficacy of FN-C/H-V on PMN accumulation in ischemic tissue, infarct size, and neurological outcomes in rats subjected to 1 hour of cerebral ischemia and 48 hours of reperfusion. RESULTS: In Experiment 1, the animals receiving FN-C/H-V at a dose of 10 to 15 mg/kg of body weight per injection showed significant reduction of PMN accumulation, reduction of infarct size, and improvement of neurological outcomes at 48 hours after reperfusion compared to untreated animals (P < 0.05). In Experiment 2, the animals receiving FN-C/H-V within 3 hours after reperfusion also showed significantly better results than untreated animals (P < 0.05). Despite the treatment delay, the administration of FN-C/H-V inhibited PMN accumulation after reperfusion but did not reduce the size of infarction when administered 6 hours after reperfusion. CONCLUSION: These data suggest that relatively late postischemic administration of FN-C/H-V is effective in brain protection after ischemia/reperfusion.

Reduction of brain injury using heparin to inhibit leukocyte accumulation in a rat model of transient focal cerebral ischemia. II. Dose-response effect and the therapeutic window.

The administration of massive doses of heparin has been demonstrated to reduce reperfusion injury. The authors have found that heparin's antileukocyte adhesion property may play a more important role than its anticoagulant property in preventing ischemia and reperfusion injury. Although the administration of massive doses of heparin has been demonstrated to reduce brain injury after ischemia and reperfusion, the optimum dosage and timing for heparin administration remain unknown. The purpose of this study was to evaluate the dose-response effect and determine the time during which heparin must be administered to inhibit leukocyte accumulation, reduce infarct size, and improve neurological outcome in rats subjected to 1 hour of cerebral ischemia and 48 hours of reperfusion. Forty-nine animals were included in the study. The animals receiving commercial unfractionated heparin at a total dose of 2.67 to 4 mg/kg showed a significant inhibition of leukocyte accumulation, reduced infarct size, and lessened neurological dysfunction 48 hours after reperfusion (p < 0.05) when compared to untreated animals. The animals receiving unfractionated heparin within 3 hours after reperfusion also showed significantly better results than untreated animals. These data indicate that standard doses of heparin prevent reperfusion injury, and relatively late postischemic administration of heparin also is effective in brain protection. These findings may have therapeutic potential as an adjunct to thrombolytic therapy and possibly for other perfusion deficiencies with leukocyte-endothelial interaction. In view of these encouraging experimental findings, the clinical application of heparin administration after ischemia and reperfusion warrants serious consideration.

Synthetic fibronectin peptides and ischemic brain injury after transient middle cerebral artery occlusion in rats.

Leukocytes play an important role in the development of ischemia-reperfusion injury. This study was conducted to ascertain whether synthetic peptides corresponding to the cell- and heparin-binding sequences of fibronectin that disturb leukocyte adhesion molecules were effective in neuronal protection after transient focal cerebral ischemia in rats. The authors evaluated the efficacy of peptides on infarction size, leukocyte infiltration in the ischemic tissue, and neurological outcome in rats subjected to 1 hour of cerebral ischemia and 48 hours of reperfusion. Twenty-one animals were divided into three groups: transient ischemia without treatment (Group I), transient ischemia with administration of vehicle (Group II), and transient ischemia with administration of fibronectin peptides (Group III). The mean myeloperoxidase activity (U/g wet wt) in the ischemic area was as follows: Group I, 0.19% +/- 0.05; Group II, 0.21% +/- 0.03; and Group III, 0.08% +/- 0.02. The mean size of the infarction as a percentage of the total hemispheric volume was as follows: Group I, 38.35% +/- 1.34%; Group II, 39.21% +/- 2.42%; and Group III, 25.81% +/- 4.87%. Group III showed a significant decrease in myeloperoxidase activity in the lesion and the infarction size was smaller when compared to Groups I and II (p < 0.05). The neurological grade in Group III was significantly better than in Groups I and II at 48 hours after reperfusion (p < 0.01). This study is the first to explore the therapeutic potential of synthetic fibronectin peptides in brain protection after transient focal ischemia, and the results also serve as a basis for studies of important cellular and molecular events that contribute to tissue damage.

Reduction of brain injury using heparin to inhibit leukocyte accumulation in a rat model of transient focal cerebral ischemia. I. Protective mechanism.

Heparin has long been established as an anticoagulant. Although heparin has been demonstrated to reduce brain injury after ischemia and reperfusion, its mechanism of action remains unknown. Recent investigations reveal that it can modulate biological processes such as binding to adhesion receptors on endothelial cells and leukocytes. The authors hypothesized that heparin's protective effect is closely related to its antileukocyte adherence property. They evaluated the efficacy of sulfated polysaccharides (unfractionated heparin, low-molecular-weight heparin, heparan sulfate, chondroitin sulfate C, and dextran sulfate) on leukocyte accumulation, infarction size, and neurological outcome after transient focal cerebral ischemia in rats subjected to 1 hour of ischemia and 48 hours of reperfusion. Forty-nine animals were included in the study. The animals receiving unfractionated heparin or dextran sulfate showed a significant reduction in leukocyte accumulation, infarct size, and neurological dysfunction 48 hours after reperfusion (p < 0.05) when compared to untreated animals. The animals receiving unfractionated heparin also showed significantly better results than the animals receiving an equivalent anticoagulant dose of low-molecular-weight heparin. These data indicate that heparin's antileukocyte property plays a more important role than its anticoagulant ability in neuronal protection. The relative potency of the sulfated polysaccharides tested in leukocyte depletion was closely related to their degree of sulfation. Thus, in addition to demonstrating the potential efficacy of heparin as a therapeutic agent for ischemia and reperfusion injury by the prevention of leukocyte accumulation, the results also serve as a basis for studying important cellular and molecular events that contribute to tissue damage.

Risk associations between HLA-DPB1 T-cell epitope matching and outcome of unrelated hematopoietic cell transplantation are independent of HLA-DPA1.

HLA-DP antigens are beta-alpha heterodimers encoded by polymorphic HLA-DPB1 and -DPA1 alleles, respectively, in strong linkage disequilibrium (LD) with each other. Non-permissive unrelated donor (UD)-recipient HLA-DPB1 mismatches across three different T-cell epitope (TCE) groups are associated with increased mortality after hematopoietic SCT (HCT), but the role of HLA-DPA1 is unclear. We studied 1281 onco-hematologic patients after 10/10 HLA-matched UD-HCT facilitated by the National Marrow Donor Program. Non-permissive mismatches defined solely by HLA-DPB1 TCE groups were associated with significantly higher risks of TRM compared to permissive mismatches (hazard ratio (HR) 1.30, confidence interval (CI) 1.06-1.53; P=0.009) or allele matches. Moreover, non-permissive HLA-DPB1 TCE group mismatches in the graft versus host (GvH) direction significantly decreased the risk of relapse compared to permissive mismatches (HR 0.55, CI 0.37-0.80; P=0.002) or allele matches. Splitting each group into HLA-DPA1*02:01 positive or negative, in frequent LD with HLA-DPB1 alleles from two of the three TCE groups, or into HLA-DPA1 matched or mismatched, did not significantly alter the observed risk associations. Our findings suggest that the effects of clinically non-permissive HLA-DPB1 TCE group mismatches are independent of HLA-DPA1, and that selection of donors with non-permissive DPB1 TCE mismatches in GvH direction might provide some protection from disease recurrence.

Optimal timing of hemodilution for brain protection in a canine model of focal cerebral ischemia.

BACKGROUND AND PURPOSE: Hemodilution 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. METHODS: Thirty-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. RESULTS: Groups 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). CONCLUSIONS: The 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.

Visualization of antigen-specific T cell activation in vivo in response to intracerebral administration of a xenopeptide.

Allogeneic or xenogenic tissues exhibit prolonged survival when grafted into the brain parenchyma in comparison to grafting into peripheral sites. The brain, therefore, has long been considered an immunologically privileged site. However, the immunological privilege of the brain is not absolute, and it cannot shield neural xenografts from rejection. In our laboratory, we are interested in determining how to prevent neural xenograft rejection. To do so, we need to first understand how the immune system responds to CNS antigens leading to graft rejection. In order to monitor immune system responses to CNS antigens an adoptive transfer system was used to directly track CNS antigen-specific CD4(+) T cell responses in vivo. This would then allow us to monitor changes in the number, activation state, and anatomic distribution of antigen-specific cells. We have found that, after intracerebral injection of xeno peptide antigens with adjuvant, antigen-specific cells accumulated in the cervical lymph node, proliferated there for several days, and then disappeared slowly from the nodes. Interestingly, peptide antigens given intracerebrally also stimulated a strong antigen-specific CD4(+) T cell response. Moreover, cells remaining in the lymph node 8 days after antigen stimulation produce IL-2 with secondary antigenic challenge. Previous studies have shown that the administration of antigens without adjuvant in a monomeric form via either the intraperitoneal or intravenous route has failed to induce cell-mediated immunity and resulted in antigen-specific T cell unresponsiveness. Our findings demonstrate that antigen delivered intracerebrally can activate immune responses in a manner different than antigen delivered to peripheral sites outside of the CNS.

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 B7-1 and B7-2, and the cell adhesion molecule ICAM-1. 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.