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BACKGROUND - Critically ill patients with acute kidney injury (AKI) can be divided into two subphenotypes, resolving or nonresolving, on the basis of the trajectory of serum creatinine. It is unknown if the biology underlying these two AKI recovery patterns is different.
METHODS - We measured eight circulating biomarkers in plasma obtained from a cohort of patients admitted to an intensive care unit (ICU) (n = 1241) with systemic inflammatory response syndrome. The biomarkers were representative of several biologic processes: apoptosis (soluble Fas), inflammation (soluble tumor necrosis factor receptor 1, interleukin 6, interleukin 8) and endothelial dysfunction, (angiopoietin 1, angiopoietin 2, and soluble vascular cell adhesion molecule 1). We tested for associations between biomarker levels and AKI subphenotypes using relative risk regression accounting for multiple hypotheses with the Bonferroni correction.
RESULTS - During the first 3 days of ICU admission, 868 (70%) subjects developed AKI; 502 (40%) had a resolving subphenotype, and 366 (29%) had a nonresolving subphenotype. Hospital mortality was 12% in the resolving subphenotype and 21% in the nonresolving subphenotype. Soluble Fas was the only biomarker associated with a nonresolving subphenotype after adjustment for age, body mass index, diabetes, and Acute Physiology and Chronic Health Evaluation III score (p = 0.005).
CONCLUSIONS - Identifying modifiable targets in the Fas-mediated pathway may lead to strategies for prevention and treatment of a clinically important form of AKI.
Idiopathic aplastic anemia (AA) is an immune-mediated and serious form of bone marrow failure. Akin to other autoimmune diseases, we have previously shown that in AA regulatory T cells (Tregs) are reduced in number and function. The aim of this study was to further characterize Treg subpopulations in AA and investigate the potential correlation between specific Treg subsets and response to immunosuppressive therapy (IST) as well as their in vitro expandability for potential clinical use. Using mass cytometry and an unbiased multidimensional analytical approach, we identified 2 specific human Treg subpopulations (Treg A and Treg B) with distinct phenotypes, gene expression, expandability, and function. Treg B predominates in IST responder patients, has a memory/activated phenotype (with higher expression of CD95, CCR4, and CD45RO within FOXP3(hi), CD127(lo) Tregs), expresses the interleukin-2 (IL-2)/STAT5 pathway and cell-cycle commitment genes. Furthermore, in vitro-expanded Tregs become functional and take on the characteristics of Treg B. Collectively, this study identifies human Treg subpopulations that can be used as predictive biomarkers for response to IST in AA and potentially other autoimmune diseases. We also show that Tregs from AA patients are IL-2-sensitive and expandable in vitro, suggesting novel therapeutic approaches such as low-dose IL-2 therapy and/or expanded autologous Tregs and meriting further exploration.
Human lung adenocarcinomas with activating mutations in EGFR (epidermal growth factor receptor) often respond to treatment with EGFR tyrosine kinase inhibitors (TKIs), but the magnitude of tumour regression is variable and transient. This heterogeneity in treatment response could result from genetic modifiers that regulate the degree to which tumour cells are dependent on mutant EGFR. Through a pooled RNA interference screen, we show that knockdown of FAS and several components of the NF-κB pathway specifically enhanced cell death induced by the EGFR TKI erlotinib in EGFR-mutant lung cancer cells. Activation of NF-κB through overexpression of c-FLIP or IKK (also known as CFLAR and IKBKB, respectively), or silencing of IκB (also known as NFKBIA), rescued EGFR-mutant lung cancer cells from EGFR TKI treatment. Genetic or pharmacologic inhibition of NF-κB enhanced erlotinib-induced apoptosis in erlotinib-sensitive and erlotinib-resistant EGFR-mutant lung cancer models. Increased expression of the NF-κB inhibitor IκB predicted for improved response and survival in EGFR-mutant lung cancer patients treated with EGFR TKI. These data identify NF-κB as a potential companion drug target, together with EGFR, in EGFR-mutant lung cancers and provide insight into the mechanisms by which tumour cells escape from oncogene dependence.
BACKGROUND - Bronchial epithelial damage and activation likely contribute to the inflammatory and airway-remodeling events characteristic of severe asthma. Interaction of Fas receptor (CD95) with its ligand (FasL; CD95L) is an important mechanism of cell-mediated apoptosis. Bronchial epithelial FasL expression provides immune barrier protection from immune cell-mediated damage.
OBJECTIVES - Membrane FasL (mFasL) is a cleavage target of matrix metalloproteinases (MMPs). We investigated whether the asthmatic T(H)2 environment might influence disease processes by increasing airway epithelial MMP-mediated cleavage of mFasL into proinflammatory soluble FasL.
METHODS - We used human airway epithelial cell lines and primary cells to model the human airway epithelium in vitro. Airway tissue from healthy subjects and patients with severe asthma was used to investigate MMP expression patterns in diseased airways.
RESULTS - We demonstrate that active MMP-7 is present in the ciliated epithelial cells of normal human airways. In patients with severe asthma, MMP-7 levels are increased in basal epithelial cells. Airway epithelial cell lines (1HAEo(-) and 16HBE14o(-)) in vitro express constitutively high levels of MMP-2 and MMP-9 but relatively low levels of MMP-7. T(H)2 cytokine (IL-4, IL-9, and IL-13) treatment of 1HAEo(-) cells increased MMP-7 mRNA and activity, triggered colocalization of intracellular MMP-7 with FasL, and caused mFasL cleavage with soluble FasL release. Small interfering RNA knockdown shows that cytokine-induced mFasL cleavage is dependent on MMP-7 activity.
CONCLUSIONS - MMPs serve multiple beneficial roles in the lung. However, chronic disordered epithelial expression of MMP-7 in patients with asthma might increase mFasL cleavage and contribute to airway epithelial damage and inflammation.
Copyright 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.
Glaucomatous optic neuropathy causes blindness through the degeneration of retinal ganglion cells (RGCs) and their axons, which comprise the optic nerve. Glaucoma traditionally is associated with elevated intraocular pressure, but often occurs or may progress with intraocular pressure in the normal range. Like other diseases of the CNS, a subset of glaucoma has been proposed to involve an autoimmune component to help explain the loss of RGCs in the absence of elevated intraocular pressure. One hypothesis involves heat shock proteins (HSPs), because increased serum levels of HSP autoantibodies are prominent in some glaucoma patients with normal pressures. In the first direct support of this hypothesis, we found that HSP27 and HSP60 immunization in the Lewis rat induced RGC degeneration and axon loss 1-4 months later in vivo in a pattern with similarities to human glaucoma, including topographic specificity of cell loss. Infiltration of increased numbers of T-cells in the retina occurred much earlier, 14-21 d after HSP immunization, and appeared to be transient. In vitro studies found that T-cells activated by HSP immunization induced RGC apoptosis via the release of the inflammatory cytokine FasL, whereas HSP immunization induced activation of microglia cells and upregulation of the FasL receptor in RGCs. In summary, our results suggest that RGC degeneration in glaucoma for selected individuals likely involves failed immunoregulation of the T-cell-RGC axis and is thus a disturbance of both proapoptotic and protective pathways.
BACKGROUND & AIMS - Although the p53 tumor suppressor has been extensively studied, many critical questions remain unanswered about the biological functions of p53 homologs, p73 and p63. Accumulating evidence suggests that both p73 and p63 play important roles in regulation of apoptosis, cell differentiation, and therapeutic drug sensitivity.
METHODS - Gastric epithelial cells were cocultured with Helicobacter pylori, and the roles of p63 and p73 proteins were assessed by luciferase reporter, real-time polymerase chain reaction, immunoblotting, and cell survival assays. Short hairpin RNA and dominant-negative mutants were used to inhibit activity of p73 and p63 isoforms. Human and murine gastric tissues were analyzed by immunohistochemistry with p73 and p63 antibodies and modified Steiner's silver method.
RESULTS - Interaction of H pylori with gastric epithelial cells leads to robust up-regulation of p73 protein in vitro and in vivo in human gastritis specimens and H pylori-infected mice. The p73 increase resulted in up-regulation of pro-apoptotic genes, NOXA, PUMA, and FAS receptor in gastric epithelial cells. Down-regulation of p73 activity suppressed cell death and Fas receptor induced by H pylori. Bacterial virulence factors within the cag pathogenicity island, c-Abl tyrosine kinase, and interaction with p63 isoforms control the activity of p73.
CONCLUSION - Our findings implicate p73 in H pylori-induced apoptosis and more generally suggest that the p53 family may play a role in the epithelial cell response to H pylori infection.
Radiolabeled monoclonal antibodies (mAb) have demonstrated measurable antitumor effects in hematologic malignancies. This outcome has been more difficult to achieve for solid tumors due, for the most part, to difficulties in delivering sufficient quantities of mAb to the tumor mass. Previous studies have shown that nonlytic levels of external beam radiation can render tumor cells more susceptible to T cell-mediated killing. The goal of these studies was to determine if the selective delivery of a radiolabeled mAb to tumors would modulate tumor cell phenotype so as to enhance vaccine-mediated T-cell killing. Here, mice transgenic for human carcinoembryonic antigen (CEA) were transplanted with a CEA expressing murine carcinoma cell line. Radioimmunotherapy consisted of yttrium-90 (Y-90)-labeled anti-CEA mAb, used either alone or in combination with vaccine therapy. A single dose of Y-90-labeled anti-CEA mAb, in combination with vaccine therapy, resulted in a statistically significant increase in survival in tumor-bearing mice over vaccine or mAb alone; this was shown to be mediated by engagement of the Fas/Fas ligand pathway. Mice receiving the combination therapy also showed a significant increase in the percentage of viable tumor-infiltrating CEA-specific CD8(+) T cells compared to vaccine alone. Mice cured of tumors demonstrated an antigen cascade resulting in CD4(+) and CD8(+) T-cell responses not only for CEA, but for p53 and gp70. These results show that systemic radiotherapy in the form of radiolabeled mAb, in combination with vaccine, promotes effective antitumor response, which may have implications in the design of future clinical trials.
Erythropoietin (Epo) is the principal regulator of the erythropoietic response to hypoxic stress, through its receptor, EpoR. The EpoR signals mediating the stress response are largely unknown, and the spectrum of progenitors that are stress responsive is not fully defined. Here, we used flow cytometry to identify stress-responsive Ter119+CD71highFSChigh early erythroblast subsets in vivo. In the mouse spleen, an erythropoietic reserve organ, early erythroblasts were present at lower frequencies and were undergoing higher rates of apoptosis than equivalent cells in bone marrow. A high proportion of splenic early erythroblasts coexpressed the death receptor Fas, and its ligand, FasL. Fas-positive early erythroblasts were significantly more likely to coexpress annexin V than equivalent, Fas-negative cells, suggesting that Fas mediates early erythroblast apoptosis in vivo. We examined several mouse models of erythropoietic stress, including erythrocytosis and beta-thalassemia. We found a dramatic increase in the frequency of splenic early erythroblasts that correlated with down-regulation of Fas and FasL from their cell surface. Further, a single injection of Epo specifically suppressed early erythroblast Fas and FasL mRNA and cell-surface expression. Therefore, Fas and FasL are negative regulators of erythropoiesis. Epo-mediated suppression of erythroblast Fas and FasL is a novel stress response pathway that facilitates erythroblast expansion in vivo.
Protein kinase B (PKBalpha/Akt1) a PI3K-dependent serine-threonine kinase, promotes T cell viability in response to many stimuli and regulates homeostasis and autoimmune disease in vivo. To dissect the mechanisms by which PKB inhibits apoptosis, we have examined the pathways downstream of PKB that promote survival after cytokine withdrawal vs Fas-mediated death. Our studies show that PKB-mediated survival after cytokine withdrawal is independent of protein synthesis and the induction of NF-kappaB. In contrast, PKB requires de novo gene transcription by NF-kappaB to block apoptosis triggered by the Fas death receptor. Using gene-deficient and transgenic mouse models, we establish that NF-kappaB1, and not c-Rel, is the critical signaling molecule downstream of the PI3K-PTEN-PKB signaling axis that regulates lymphocyte homeostasis.
Inducible protection from apoptosis in vivo controls the size of cell populations. An important question in this respect is how differentiation affects mechanisms of apoptosis regulation. Among mature T lymphocytes, the NF-kappaB/Rel transcription factors are coupled to receptors that control cell population sizes by concurrently regulating survival and multiplication. In the present study, we used a transgenic inhibitor of NF-kappaB/Rel signaling to investigate the role of this pathway in proliferation and death of mature T cells in vivo. The results indicate that NF-kappaB integrates two critical yet distinct molecular pathways preventing apoptosis affected by the death receptor Fas, coordinately regulating levels of FLIP and Bcl-x(L) in primary T cells. Surprisingly, NF-kappaB blockade preferentially impacted naive as compared to memory T cells. The Fas/FasL pathway was linked to these findings by evidence that the abnormalities imposed by NF-kappaB inhibition were ameliorated by Fas deficiency, particularly for the CD4(+) lineage. Moreover, levels of an inhibitor of Fas-mediated apoptosis, c-FLIP, were diminished in cells expressing the transgenic inhibitor. NF-kappaB was also linked to T cell survival in vivo by mediating induction of Bcl-x(L): restoration of Bcl-x(L) levels reversed the preferential deficit of naive T cells, differentially impacting the CD4 and CD8 subsets. These results show that promoting survival and effective multiplication are central roles for NF-kappaB in T lymphoid homeostasis in vivo, but this effect and its underlying mechanisms are influenced by the developmental state of the lymphocyte.