Biomarkers of immunotherapy in glioblastoma.

Glioblastoma (GBM) is the most common primary brain cancer, comprising half of all malignant brain tumors. Patients with GBM have a poor prognosis, with a median survival of 14-15 months. Current therapies for GBM, including chemotherapy, radiotherapy, and surgical resection, remain inadequate. Novel therapies are required to extend patient survival. Although immunotherapy has shown promise in other cancers, including melanoma and non-small lung cancer, its efficacy in GBM has been limited to subsets of patients. Identifying biomarkers of immunotherapy response in GBM could help stratify patients, identify new therapeutic targets, and develop more effective treatments. This article reviews existing and emerging biomarkers of clinical response to immunotherapy in GBM. The scope of this review includes immune checkpoint inhibitor and antitumoral vaccination approaches, summarizing the variety of molecular, cellular, and computational methodologies that have been explored in the setting of anti-GBM immunotherapies.

Biologically-informed deep neural networks provide quantitative assessment of intratumoral heterogeneity in post-treatment glioblastoma.

Intratumoral heterogeneity poses a significant challenge to the diagnosis and treatment of glioblastoma (GBM). This heterogeneity is further exacerbated during GBM recurrence, as treatment-induced reactive changes produce additional intratumoral heterogeneity that is ambiguous to differentiate on clinical imaging. There is an urgent need to develop non-invasive approaches to map the heterogeneous landscape of histopathological alterations throughout the entire lesion for each patient. We propose to predictively fuse Magnetic Resonance Imaging (MRI) with the underlying intratumoral heterogeneity in recurrent GBM using machine learning (ML) by leveraging image-localized biopsies with their associated locoregional MRI features. To this end, we develop BioNet, a biologically-informed neural network model, to predict regional distributions of three tissue-specific gene modules: proliferating tumor, reactive/inflammatory cells, and infiltrated brain tissue. BioNet offers valuable insights into the integration of multiple implicit and qualitative biological domain knowledge, which are challenging to describe in mathematical formulations. BioNet performs significantly better than a range of existing methods on cross-validation and blind test datasets. Voxel-level prediction maps of the gene modules by BioNet help reveal intratumoral heterogeneity, which can improve surgical targeting of confirmatory biopsies and evaluation of neuro-oncological treatment effectiveness. The non-invasive nature of the approach can potentially facilitate regular monitoring of the gene modules over time, and making timely therapeutic adjustment. These results also highlight the emerging role of ML in precision medicine.

Convection-enhanced delivery of immunomodulatory therapy for high-grade glioma.

The prognosis for glioblastoma has remained poor despite multimodal standard of care treatment, including temozolomide, radiation, and surgical resection. Further, the addition of immunotherapies, while promising in a number of other solid tumors, has overwhelmingly failed in the treatment of gliomas, in part due to the immunosuppressive microenvironment and poor drug penetrance to the brain. Local delivery of immunomodulatory therapies circumvents some of these challenges and has led to long-term remission in select patients. Many of these approaches utilize convection-enhanced delivery (CED) for immunological drug delivery, allowing high doses to be delivered directly to the brain parenchyma, avoiding systemic toxicity. Here, we review the literature encompassing immunotherapies delivered via CED-from preclinical model systems to clinical trials-and explore how their unique combination elicits an antitumor response by the immune system, decreases toxicity, and improves survival among select high-grade glioma patients.

Human stem cell-derived thymic epithelial cells enhance human T-cell development in a xenogeneic thymus.

BACKGROUND: Generation of thymic tissue from pluripotent stem cells would provide therapies for acquired and congenital thymic insufficiency states. OBJECTIVES: This study aimed to generate human thymic epithelial progenitors from human embryonic stem cells (hES-TEPs) and to assess their thymopoietic function in vivo. METHODS: This study differentiated hES-TEPs by mimicking developmental queues with FGF8, retinoic acid, SHH, Noggin, and BMP4. Their function was assessed in reaggregate cellular grafts under the kidney capsule and in hybrid thymi by incorporating them into swine thymus (SwTHY) grafts implanted under the kidney capsules of immunodeficient mice that received human hematopoietic stem and progenitor cells (hHSPCs) intravenously. RESULTS: Cultured hES-TEPs expressed FOXN1 and formed colonies expressing EPCAM and both cortical and medullary thymic epithelial cell markers. In thymectomized immunodeficient mice receiving hHSPCs, hES-TEPs mixed with human thymic mesenchymal cells supported human T-cell development. Hypothesizing that support from non-epithelial thymic cells might allow long-term function of hES-TEPs, the investigators injected them into SwTHY tissue, which supports human thymopoiesis in NOD severe combined immunodeficiency IL2Rγnull mice receiving hHSPCs. hES-TEPs integrated into SwTHY grafts, enhanced human thymopoiesis, and increased peripheral CD4+ naive T-cell reconstitution. CONCLUSIONS: This study has developed and demonstrated in vivo thymopoietic function of hES-TEPs generated with a novel differentiation protocol. The SwTHY hybrid thymus model demonstrates beneficial effects on human thymocyte development of hES-TEPs maturing in the context of a supportive thymic structure.

Probing tissue transglutaminase mediated vascular smooth muscle cell aging using a novel transamidation-deficient Tgm2-C277S mouse model.

Tissue transglutaminase (TG2), a multifunctional protein of the transglutaminase family, has putative transamidation-independent functions in aging-associated vascular stiffening and dysfunction. Developing preclinical models will be critical to fully understand the physiologic relevance of TG2's transamidation-independent activity and to identify the specific function of TG2 for therapeutic targeting. Therefore, in this study, we harnessed CRISPR-Cas9 gene editing technology to introduce a mutation at cysteine 277 in the active site of the mouse Tgm2 gene. Heterozygous and homozygous Tgm2-C277S mice were phenotypically normal and were born at the expected Mendelian frequency. TG2 protein was ubiquitously expressed in the Tgm2-C277S mice at levels similar to those of wild-type (WT) mice. In the Tgm2-C277S mice, TG2 transglutaminase function was successfully obliterated, but the transamidation-independent functions ascribed to GTP, fibronectin, and integrin binding were preserved. In vitro, a remodeling stimulus led to the significant loss of vascular compliance in WT mice, but not in the Tgm2-C277S or TG2-/- mice. Vascular stiffness increased with age in WT mice, as measured by pulse-wave velocity and tensile testing. Tgm2-C277S mice were protected from age-associated vascular stiffening, and TG2 knockout yielded further protection. Together, these studies show that TG2 contributes significantly to overall vascular modulus and vasoreactivity independent of its transamidation function, but that transamidation activity is a significant cause of vascular matrix stiffening during aging. Finally, the Tgm2-C277S mice can be used for in vivo studies to explore the transamidation-independent roles of TG2 in physiology and pathophysiology.

Efficacy and immunologic effects of extracorporeal photopheresis plus interleukin-2 in chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) affects >50% of hematopoietic stem cell transplant patients. Extracorporeal photopheresis (ECP), an immunomodulatory therapy, provides clinical benefit in steroid-refractory (SR) cGVHD, possibly via regulatory T (Treg) and natural killer (NK) cell expansion. We demonstrated that low-dose interleukin-2 (IL2) led to clinical improvement in SR-cGVHD and stimulated preferential Treg and NK-cell expansion with minimal effect on conventional T (Tcon) cells. We evaluated the effect of ECP (weeks 1-16) plus IL2 (1 × 106 IU/m2, weeks 9-16) in 25 adult patients with SR-cGVHD in a prospective phase 2 trial. Objective responses occurred in 29% and 62% of evaluable patients at weeks 8 (ECP alone) and 16 (ECP plus IL2), respectively. Eight weeks of ECP alone was associated with a marked decline in CD4+ Tcon (P = .03) and CD8+ T cells (P = .0002), with minimal change in Treg cells, Treg:Tcon cell ratio, or NK cells. Adding IL2 induced an increase in Treg cells (P < .05 at weeks 9-16 vs week 8), Treg:Tcon cell ratio (P < .0001 at weeks 9-16 vs week 8), and NK cells (P < .05 at weeks 9-16 vs week 8). Patients responding to ECP alone had significantly fewer CD4+ Tcon and CD8+ T cells at baseline compared with patients who responded after IL2 addition and patients who did not respond; neither Treg nor NK cells were associated with response to ECP alone. Altogether, ECP plus IL2 is safe and effective in patients with SR-cGVHD. ECP and IL2 have distinct immunologic effects, suggesting different therapeutic mechanisms of action. This trial was registered at www.clinicaltrials.gov as #NCT02340676.

Transfusion Reactions.

Transfusion reactions are common occurrences, and clinicians who order or transfuse blood components need to be able to recognize adverse sequelae of transfusion. The differential diagnosis of any untoward clinical event should always consider adverse sequelae of transfusion, even when transfusion occurred weeks earlier. There is no pathognomonic sign or symptom that differentiates a transfusion reaction from other potential medical problems, so vigilance is required during and after transfusion when a patient presents with a change in clinical status. This review covers the presentation, mechanisms, and management of transfusion reactions that are commonly encountered, and those that can be life-threatening.

Hemolysis from ABO Incompatibility.

ABO incompatibility of red blood cells leads to brisk complement-mediated lysis, particularly in the setting of red cell transfusion. The ABO blood group is the most clinically significant blood group because of preformed immunoglobulin M (IgM) and IgG antibodies to ABO blood group antigens (isohemagglutinins) in everyone except group AB individuals. In addition to transfusion, ABO incompatibility can cause hemolysis in hematopoietic and solid organ transplantation, hemolytic disease of the newborn, and intravenous immunoglobulin infusion. It is important to prevent ABO incompatibility when possible and to anticipate complications when ABO incompatibility is unavoidable.

Length of red cell unit storage and risk for delirium after cardiac surgery.

BACKGROUND: The time that red cell units are stored before transfusion may be associated with postoperative complications, although the evidence is conflicting. However, the association between the length of red cell unit storage and postoperative delirium has not been explored. We hypothesized that the length of storage of transfused red cell units would be associated with delirium after cardiac surgery. METHODS: We conducted a case-control study in which patients undergoing coronary artery bypass, valve, or ascending aorta surgery with cardiopulmonary bypass at Johns Hopkins from 2005 to 2011 were eligible for inclusion. Patients were excluded if they did not receive red cell units, received >4 red cell units during hospitalization, received any transfusion after the first postoperative day, or received red cell units that were not exclusively stored for ≤14 days or >14 days. Eighty-seven patients met transfusion-related inclusion criteria and developed postoperative delirium. Controls who did not develop delirium were selected from the same source population of eligible patients and were matched 1:1 based on age (± 5 years), 2- to 2.5-year band of date of surgery, and surgical procedure. For each patient, we calculated the average storage duration of all transfused red cell units. The primary outcome was odds of delirium in patients who were transfused red cell units with exclusive storage duration >14 days compared with that of ≤14 days. Secondary outcomes were odds of delirium with each increasing day of average red cell unit storage duration. We used conditional multivariable regression to test our hypotheses. RESULTS: In conditional multivariable analysis of 87 case-control pairs, there was no difference in the odds of patients developing delirium if they were transfused red cell units with an exclusive storage age >14 days compared with that ≤14 days (odds ratio [OR] 1.83; 95% confidence interval, 0.73-4.58, P = 0.20). Each additional day of average red cell unit storage beyond 14 days was associated with a 1.01- to 1.13-fold increase in the odds of postoperative delirium (OR, 1.07; P = 0.03). Each additional day of average storage beyond 21 days was associated with a 1.02- to 1.23-fold increase in the odds of postoperative delirium (OR, 1.12; P = 0.02). CONCLUSIONS: Transfusion of red cell units that have been stored for >14 days is not associated with increased odds of delirium. However, each additional day of storage >14 or 21 days may be associated with increased odds of postoperative delirium in patients undergoing cardiac surgery. More research is needed to further characterize the association between delirium and storage duration of transfused red cell units.

Odds of transfusion for older adults compared to younger adults undergoing surgery.

BACKGROUND: Recent randomized controlled trials have shown no benefit for transfusion to a hemoglobin >10 g/dL compared with lower hemoglobin thresholds in the perioperative period, even among older adults. Nevertheless, physicians may choose to transfuse older adults more liberally than younger adults. It is unclear whether older patients have higher odds than younger patients of being transfused in the perioperative period. Our objective in this study was to determine whether the odds of transfusion are higher in older patients than in younger patients in the perioperative period. METHODS: We conducted this retrospective observational cohort study at a tertiary care academic medical center. We included adults who had undergone a surgical procedure as an inpatient at our institution from January 2010 to February 2012. The primary analysis compared the odds of transfusion for patients >65 years old with the odds of transfusion in younger patients based on multilevel multivariable logistic regression analyses including adjustment for comorbidities, surgical service, lowest in-hospital hemoglobin value, gender, and estimated surgical blood loss and accounted for clustering by the surgeon and procedure. RESULTS: We included 20,930 patients in this analysis. In multilevel models adjusted for comorbidities, surgical service, estimated surgical blood loss, and lowest in-hospital hemoglobin value, with surgeon and procedure as random effects, patients > 65 years old had 62% greater odds (odds ratio, 1.62; 95% confidence interval, 1.40-1.88; P < 0.0001) of being transfused than did younger patients. When patients were stratified by lowest in-hospital hemoglobin (7.00-7.99, 8.00-8.99, 9.00-9.99, and ≥10.00 g/dL), the odds of transfusion generally increased with each additional decade of age in every stratum, except for that containing patients in whom the lowest in-hospital hemoglobin did not decrease below 10 g/dL. When the odds of transfusion were compared between younger and older patients, significant differences were observed among surgical services (P = 0.02) but not among anesthesia specialty divisions (P = 0.9). CONCLUSIONS: Older adults have greater odds of receiving red blood cell transfusion in the perioperative period than do younger patients, despite the lack of evidence supporting higher hemoglobin triggers in elderly patients. Further research is needed to determine whether transfusion practice in the elderly is an opportunity for education to improve blood management.

Partial manual exchange reduces iron accumulation during chronic red cell transfusions for sickle cell disease.

Iron overload is an inevitable consequence of chronic red cell transfusions without erythrocytapheresis or chelation therapy. The effectiveness of partial manual exchange, a technique used to slow iron loading, has not been evaluated. We evaluated all children with sickle cell disease (SCD) receiving chronic transfusion to identify chelation-naive subjects who had quantitative liver iron concentration (LIC) studies. Seventeen chelation-naive children with SCD received a median of 29 transfusions before first LIC determination. Serum ferritin concentrations were assessed before each transfusion. The mean volume of blood phlebotomized before each transfusion was 5.1±1.8 mL/kg, which cumulatively resulted in a calculated median of 35.0 mg/kg iron removal. Using linear regression, pretransfusion phlebotomy resulted in a statistically significant reduction in ferritin (-8.8 ng/mL of ferritin for each mg/kg of iron phlebotomized, P=0.02). A reduction in LIC from pretransfusion phlebotomy could not be established (P=0.4). Partial manual exchanges appear to be an effective strategy for slowing the pace of iron loading in the setting of chronic transfusion for SCD.

Hemoglobin drift after cardiac surgery.

BACKGROUND: Recent literature suggests that a restrictive approach to red blood cell transfusions is associated with improved outcomes in cardiac surgery patients. Even in the absence of bleeding, intravascular fluid shifts cause hemoglobin levels to drift postoperatively, possibly confounding the decision to transfuse. The purpose of this study was to define the natural progression of hemoglobin levels in postoperative cardiac surgery patients. METHODS: All cardiac surgery patients from October 2010 through March 2011 who did not receive a postoperative transfusion were included. Primary stratification was by intraoperative transfusion status. Change in hemoglobin was evaluated relative to the initial postoperative hemoglobin. Maximal drift was defined as the maximum minus the minimum hemoglobin for a given hospitalization. Final drift was defined as the difference between initial and discharge hemoglobin. RESULTS: The final cohort included 199 patients: 71 (36%) received an intraoperative transfusion, whereas 128 (64%) did not. The average initial and final hemoglobin levels for all patients were 11.0±1.4 g/dL and 9.9±1.3 g/dL, respectively, giving a final drift of 1.1±1.4 g/dL. The maximal drift was 1.8±1.1 g/dL and was similar regardless of intraoperative transfusion status (p=0.9). Although all patients' hemoglobin initially dropped, 79% of patients reached a nadir and experienced a mean recovery of 0.7±0.7 g/dL by discharge. On multivariable analysis, increasing cardiopulmonary bypass time was significantly associated with total hemoglobin drift (coefficient/hour, 0.3 [0.1-0.5] g/dL; p=0.02). CONCLUSIONS: In this report of hemoglobin drift after cardiac surgery, although all postoperative patients experienced downward hemoglobin drift, 79% of patients exhibited hemoglobin recovery before discharge. Physicians should consider the eventual upward hemoglobin drift before administering red blood cell transfusions.

Bleeding risks are higher in children versus adults given prophylactic platelet transfusions for treatment-induced hypoproliferative thrombocytopenia.

Age-group analyses were conducted of patients in the prophylactic platelet dose trial (PLADO), which evaluated the relation between platelet dose per transfusion and bleeding. Hospitalized patients with treatment-induced hypoproliferative thrombocytopenia were randomly assigned to 1 of 3 platelet doses: 1.1 × 10(11), 2.2 × 10(11), or 4.4 × 10(11) platelets/m(2) per transfusion, given for morning counts of ≤ 10 000 platelets/µL. Daily hemostatic assessments were performed. The primary end point (percentage of patients who developed grade 2 or higher World Health Organization bleeding) was evaluated in 198 children (0-18 years) and 1044 adults. Although platelet dose did not predict bleeding for any age group, children overall had a significantly higher risk of grade 2 or higher bleeding than adults (86%, 88%, 77% vs 67% of patients aged 0-5 years, 6-12 years, 13-18 years, vs adults, respectively) and more days with grade 2 or higher bleeding (median, 3 days in each pediatric group vs 1 day in adults; P < .001). The effect of age on bleeding differed by disease treatment category and was most pronounced among autologous transplant recipients. Pediatric subjects were at higher risk of bleeding over a wide range of platelet counts, indicating that their excess bleeding risk may be because of factors other than platelet counts.

Pre- and post-operative plasma glial fibrillary acidic protein levels in patients with newly diagnosed gliomas.

Therapies that disrupt or repair blood-brain barrier integrity can result in major changes in MRI images even when the tumor volume remains constant. Thus, a reliable blood-based tumor biomarker could significantly improve clinical care and research studies in these patients. This study was performed to assess plasma concentrations of glial fibrillary acidic protein (GFAP) in patients with high- and low-grade gliomas before and after debulking surgery. Pre-operative plasma was collected from 33 patients with radiation- and chemotherapy-naïve gliomas. Additional plasma was collected 24-48 h post-operatively from 23 of these patients. Plasma GFAP (pGFAP) concentrations were determined using an electrochemiluminescent immunoassay and were analyzed as a function of tumor grade, tumor GFAP expression, the integrity of the blood-brain barrier, and post-operative status. Detectable pGFAP levels (≥ 0.04 ng/mL) were found pre-operatively in 52 % of patients and post-operatively in 96 %. Detectable pGFAP was more common in patients with WHO grade IV (100 %) than WHO grade III (56 %) or WHO grade II gliomas (20 %). No patient with undetectable GFAP had WHO grade IV glioma. Higher pGFAP concentrations were also associated with contrast enhancement but not related to tumor GFAP expression. GFAP is commonly detected in the plasma of patients with high-grade gliomas. pGFAP levels rise rather than fall following debulking surgery which is probably a result of surgical trauma. GFAP remains a potentially informative plasma biomarker for gliomas. Longitudinal studies are required to correlate pGFAP levels with patient outcomes.

Allergic agonists in apheresis platelet products are associated with allergic transfusion reactions.

BACKGROUND: The mechanisms that underlie allergic transfusion reactions (ATRs) are not well characterized, but likely involve recipient, donor, and product factors. To assess product factors associated with ATRs, we investigated candidate mediators in apheresis platelet (PLT) products associated with ATRs and controls. STUDY DESIGN AND METHODS: Using bead-based and standard enzyme-linked immunosorbent assays, we tested supernatants from 20 consecutive apheresis PLT transfusions associated with ATRs and 30 control products for concentrations of mediators in three categories: acute inflammatory mediators, direct agonists of basophils and mast cells, and growth and/or priming factors of basophils and mast cells. RESULTS: Median concentrations of the direct allergic agonists C5a, brain-derived neurotrophic factor (BDNF), and CCL5 (RANTES) were 16.6, 41.8, and 13.9% higher, respectively, in the supernatant of apheresis PLT products that were most strongly associated with ATRs (p < 0.05 for each mediator). Other direct agonists (macrophage inflammatory protein-1α, monocyte chemotactic protein-1, eotaxin-1, interleukin-8) were similar between groups. Concentrations of acute inflammatory mediators and basophil growth and/or priming factors were also similar between groups (p > 0.2 for all associations). CONCLUSION: The allergic agonists C5a, BDNF, and CCL5 may be mediators of ATRs in apheresis PLT products. Acute inflammatory proteins and basophil and/or mast cell growth and priming factors do not appear to be associated with apheresis PLT products that cause ATRs.

Regulation of seed germination in the close Arabidopsis relative Lepidium sativum: a global tissue-specific transcript analysis.

The completion of germination in Lepidium sativum and other endospermic seeds (e.g. Arabidopsis [Arabidopsis thaliana]) is regulated by two opposing forces, the growth potential of the radicle (RAD) and the resistance to this growth from the micropylar endosperm cap (CAP) surrounding it. We show by puncture force measurement that the CAP progressively weakens during germination, and we have conducted a time-course transcript analysis of RAD and CAP tissues throughout this process. We have also used specific inhibitors to investigate the importance of transcription, translation, and posttranslation levels of regulation of endosperm weakening in isolated CAPs. Although the impact of inhibiting translation is greater, both transcription and translation are required for the completion of endosperm weakening in the whole seed population. The majority of genes expressed during this process occur in both tissues, but where they are uniquely expressed, or significantly differentially expressed between tissues, this relates to the functions of the RAD as growing tissue and the CAP as a regulator of germination through weakening. More detailed analysis showed that putative orthologs of cell wall-remodeling genes are expressed in a complex manner during CAP weakening, suggesting distinct roles in the RAD and CAP. Expression patterns are also consistent with the CAP being a receptor for environmental signals influencing germination. Inhibitors of the aspartic, serine, and cysteine proteases reduced the number of isolated CAPs in which weakening developed, and inhibition of the 26S proteasome resulted in its complete cessation. This indicates that targeted protein degradation is a major control point for endosperm weakening.

Multicenter survey on the outcome of transplantation of hematopoietic cells in patients with the complete form of DiGeorge anomaly.

Seventeen patients transplanted with hematopoietic cells to correct severe T lymphocyte immunodeficiency resulting from complete DiGeorge anomaly were identified worldwide, and retrospective data were obtained using a questionnaire-based survey. Patients were treated at a median age of 5 months (range, 2-53 months) between 1995 and 2006. Bone marrow was used in 11 procedures in 9 cases: 6 from matched unrelated donors, 4 from human leukocyte antigen (HLA)-identical siblings, and one haploidentical parent with T-cell depletion. Unmobilized peripheral blood was used in 8 cases: 5 from HLA-identical siblings, one from a matched unrelated donor, one from an HLA-identical parent, and one unrelated matched cord blood. Conditioning was used in 5 patients and graft-versus-host disease prophylaxis in 11 patients. Significant graft-versus-host disease occurred in 9 patients, becoming chronic in 3. Median length of follow-up was 13 months, with transplantation from HLA-matched sibling showing the best results. Median survival among deceased patients (10 patients) was 7 months after transplantation (range, 2-18 months). The overall survival rate was 41%, with a median follow-up of 5.8 years (range, 4-11.5 years). Among survivors, median CD3 and CD4 counts were 806 (range, 644-1224) and 348 (range, 225-782) cells/mm(3), respectively, CD4(+)/CD45RA(+) cells remained very low, whereas mitogen responses were normalized.

Ethylene interacts with abscisic acid to regulate endosperm rupture during germination: a comparative approach using Lepidium sativum and Arabidopsis thaliana.

The micropylar endosperm cap covering the radicle in the mature seeds of most angiosperms acts as a constraint that regulates seed germination. Here, we report on a comparative seed biology study with the close Brassicaceae relatives Lepidium sativum and Arabidopsis thaliana showing that ethylene biosynthesis and signaling regulate seed germination by a mechanism that requires the coordinated action of the radicle and the endosperm cap. The larger seed size of Lepidium allows direct tissue-specific biomechanical, biochemical, and transcriptome analyses. We show that ethylene promotes endosperm cap weakening of Lepidium and endosperm rupture of both species and that it counteracts the inhibitory action of abscisic acid (ABA) on these two processes. Cross-species microarrays of the Lepidium micropylar endosperm cap and the radicle show that the ethylene-ABA antagonism involves both tissues and has the micropylar endosperm cap as a major target. Ethylene counteracts the ABA-induced inhibition without affecting seed ABA levels. The Arabidopsis loss-of-function mutants ACC oxidase2 (aco2; ethylene biosynthesis) and constitutive triple response1 (ethylene signaling) are impaired in the 1-aminocyclopropane-1-carboxylic acid (ACC)-mediated reversion of the ABA-induced inhibition of seed germination. Ethylene production by the ACC oxidase orthologs Lepidium ACO2 and Arabidopsis ACO2 appears to be a key regulatory step. Endosperm cap weakening and rupture are promoted by ethylene and inhibited by ABA to regulate germination in a process conserved across the Brassicaceae.

Glycosylphosphatidylinositol-anchored protein deficiency confers resistance to apoptosis in PNH.

OBJECTIVE: Investigate the contribution of PIG-A mutations to clonal expansion in paroxysmal nocturnal hemoglobinuria (PNH). MATERIALS AND METHODS: Primary CD34+ hematopoietic progenitors from PNH patients were assayed for annexin-V positivity by flow cytometry in a cell-mediated killing assay using autologous effectors from PNH patients or allogeneic effectors from healthy controls. To specifically assess the role of the PIG-A mutation in the development of clonal dominance and address confounders of secondary mutation and differential immune attack that can confound experiments using primary cells, we established an inducible PIG-A CD34+ myeloid cell line, TF-1. Apoptosis resistance was assessed after exposure to allogeneic effectors, NK92 cells (an interleukin-2-dependent cell line with the phenotype and function of activated natural killer [NK] cells), tumor necrosis factor (TNF)-alpha, and gamma-irradiation. Apoptosis was measured by annexin-V staining and caspase 3/7 activity. RESULTS: In PNH patients, CD34+ hematopoietic progenitors lacking glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-AP(-)) were less susceptible than GPI-AP+ CD34+ precursors to autologous (8% vs 49%; p < 0.05) and allogeneic (28% vs 58%; p < 0.05) cell-mediated killing from the same patients. In the inducible PIG-A model, GPI-AP(-) TF-1 cells exhibited less apoptosis than induced, GPI-AP+ TF-1 cells in response to allogeneic cell-mediated killing, NK92-mediated killing, TNF-alpha, and gamma-irradiation. GPI-AP(-) TF-1 cells maintained resistance to apoptosis when effectors were raised against GPI-AP(-) cells, arguing against a GPI-AP being the target of immune attack in PNH. NK92-mediated killing was partially inhibited with blockade by specific antibodies to the stress-inducible GPI-AP ULBP1 and ULBP2 that activate immune effectors. Clonal competition experiments demonstrate that the mutant clone expands over time under proapoptotic conditions with TNF-alpha. CONCLUSION: PIG-A mutations contribute to clonal expansion in PNH by conferring a survival advantage to hematopoietic progenitors under proapoptotic stresses.