Sustained effector function of IL-12/15/18–preactivated NK cells against established tumors

Natural killer cell (NK cell)–based immunotherapy of cancer is hampered by the transient effector function of NK cells. Recently, mouse IL-12/15/18–preactivated NK cells were shown to persist with sustained effector function in vivo. Our study investigated the antitumor activity of such NK cells. A single injection of syngeneic IL-12/15/18–preactivated NK cells, but neither naive nor IL-15– or IL-2–pretreated NK cells, combined with irradiation substantially reduced growth of established mouse tumors. Radiation therapy (RT) was essential for the antitumor activity of transferred NK cells. IL-12/15/18–preactivated NK cells expressed high levels of IL-2Rα (CD25), and their rapid in vivo proliferation depended on IL-2 produced by CD4⁺ T cells. IL-12/15/18–preactivated NK cells accumulated in the tumor tissue and persisted at high cell numbers with potent effector function that required the presence of CD4⁺ T cells. RT greatly increased numbers and function of transferred NK cells. Human IL-12/15/18–preactivated NK cells also displayed sustained effector function in vitro. Our study provides a better understanding for the rational design of immunotherapies of cancer that incorporate NK cells. Moreover, our results reveal an essential role of CD4⁺ T cell help for sustained antitumor activity by NK cells linking adaptive and innate immunity.NK cells are potent antitumor effector cells (Cerwenka and Lanier, 2001; Ljunggren and Malmberg, 2007; Terme et al., 2008; Vivier et al., 2008). Accordingly, individuals with low NK cell activity display an increased risk to develop cancer (Imai et al., 2000), and high numbers of intratumoral NK cells are often correlated with improved prognosis for cancer patients (Coca et al., 1997; Villegas et al., 2002). Human tumors frequently express low levels of MHC class I molecules that interact with inhibitory NK cell receptors. For instance, alterations in the β2m gene can lead to an almost complete and irreversible lack of MHC class I in melanoma cells (D’Urso et al., 1991). In addition, many tumor cells express high levels of ligands for activating NK cell receptors (Raulet and Guerra, 2009), leading to efficient recognition by NK cells (Vivier et al., 2008; Pegram et al., 2011). So far, NK cell–based therapy was mainly successful in patients suffering from leukemia (Moretta et al., 2011). Acute myeloid leukemia patients that received haploidentical bone marrow grafts from Killer immunoglobulin receptor (KIR)–mismatched donors displayed a significantly increased 5-yr disease-free survival (Ruggeri et al., 2002). In addition, clinical benefits were observed upon infusion of KIR-mismatched NK cells after stem cell transplantation (Passweg et al., 2004; Miller et al., 2005; Geller and Miller, 2011; Geller et al., 2011). However, adoptive transfer of autologous IL-2–activated NK cells in patients suffering from solid tumors such as melanoma or renal cell carcinoma did not result in clinical benefits (Parkhurst et al., 2011). Thus, novel strategies are urgently needed to improve the antitumor activity of transferred NK cells against solid tumors.During certain viral infections (Sun et al., 2009a) and contact hypersensitivity reactions (O’Leary et al., 2006), persistent NK cell subpopulations mounting recall responses were detected, indicating previously unappreciated memory properties of NK cells (Paust and von Andrian, 2011; Sun et al., 2011; Vivier et al., 2011). In addition, NK cells preactivated with IL-12, IL-15, and IL-18 in vitro for 15 h were detectable at high numbers 3 wk after transfer into RAG-1^−/− mice and produced high levels of IFN-γ upon restimulation (Cooper et al., 2009). Much lower cell numbers and IFN-γ production were observed when IL-15–preactivated NK cells were transferred. Thus, the activation of NK cells with certain cytokines resulted in an NK cell population with enhanced effector function upon restimulation, indicating that NK cells are able to retain memory of prior activation.Because IL-12/15/18–preactivated NK cells were shown to persist with sustained effector function after restimulation (Cooper et al., 2009), we investigated whether application of IL-12/15/18–preactivated NK cells improves current protocols of immunotherapy of cancer. Our study reveals that a single injection of IL-12/15/18–preactivated NK cells, but neither naive nor of IL-15– or IL-2–pretreated NK cells, combined with radiation therapy (RT), substantially reduced growth of established mouse tumors. Our results raise the possibilities for the development of novel NK cell–based therapeutic strategies for clinical application.     

High lactate dehydrogenase levels at admission for painful vaso-occlusive crisis is associated with severe outcome in adult SCD patients

Objectives

The aim of this study is to assess biological prognostic factors at the onset of vaso-occlusive crisis (VOC) in adults with sickle cell disease (SCD).

Methods

A monocentric prospective study including all patients admitted for VOC in a reference center for SCD was utilized. We used multivariate logistic regression to find independent predictors of severe evolution, defined by death or a worsening clinical state indicating transfusion or transfer to the intensive care unit.

Results

Eighty eight patients were included, 63% were women, median age of 23 years, and 90% of patients were homozygous SCD, 10% compound heterozygous. VOC became severe in 17 patients. Patients with severe VOC were more frequently males, who also had higher white blood cell (WBC) count, procalcitonin (PCT), and lactate dehydrogenase (LDH) levels. LDH level was the best predictor of the outcome; WBC and PCT had no significant added predictive values when coupled with LDH in multivariable models, even in patients with fever or acute chest syndrome. Severe evolution always occurred when LDH levels were over 4 times the upper limit of the normal range at admission and never occurred when LDH levels were within the normal range.

Conclusion

Further studies should confirm the predictive value of LDH before its widespread use as a prognostic factor. If it is confirmed, the benefit of preemptive transfusion when LDH levels at admission are very high could be investigated.     

Beta cell function: the role of macrophage migration inhibitory factor

During evolution, beta cells adapted to a sole aim: the production and stimulus-dependent secretion of insulin. This acquired specificity was accompanied by a loss of protection mechanisms predisposing beta cell to a high vulnerability. Among beta cell-damaging molecules, a new one has been identified recently: macrophage migration inhibitory factor (MIF). MIF was at first designated as a T-cell product that inhibits random movement of macrophages. Over the years, the number of functions attributed to this protein increased significantly, positioning MIF at the top of inflammatory cascade in the combat against infection and in immunoinflammatory and autoimmune diseases. This exceptionally versatile molecule regulates insulin secretion in physiological conditions, while in pathological states it alters beta cell function and induces their apoptosis or necrosis and affects beta cell neoplasia.     

Macrophage migration inhibitory factor deficiency protects pancreatic islets from cytokine-induced apoptosis in vitro

During pathogenesis of diabetes, pancreatic islets are exposed to high levels of cytokines and other inflammatory mediators that induce deterioration of insulin-producing beta cells. Macrophage migration inhibitory factor (MIF) plays a key role in the onset and development of several immunoinflammatory diseases and also controls apoptotic cell death. Because the occurrence of apoptosis plays a pathogenetic role in beta cell death during type 1 diabetes development and MIF is expressed in beta cells, we explored the influence of MIF deficiency on cytokine-induced apoptosis in pancreatic islets. The results indicated clearly that elevated MIF secretion preceded C57BL/6 pancreatic islets death induced by interferon (IFN)-γ + tumour necrosis factor (TNF)-α + interleukin (IL)-1β. Consequently, MIF-deficient [MIF-knock-out (KO)] pancreatic islets or islet cells showed significant resistance to cytokine-induced death than those isolated from C57BL/6 mice. Furthermore, upon exposure to cytokines pancreatic islets from MIF-KO mice maintained normal insulin expression and produced less cyclooxygenase-2 (COX-2) than those from wild-type C57BL6 mice. The final outcome of cytokine-induced islet apoptosis in islets from wild-type mice was the activation of mitochondrial membrane pore-forming protein Bcl-2-associated X protein and effector caspase 3. In contrast, these apoptotic mediators remained at normal levels in islets from MIF-KO mice suggesting that MIF absence prevented initiation of the mitochondrial apoptotic pathway. Additionally, the protection from apoptosis was also mediated by up-regulation of prosurvival kinase extracellular-regulated kinase 1/2 in MIF-KO islets. These data indicate that MIF is involved in the propagation of pancreatic islets apoptosis probably via nuclear factor-κB and mitochondria-related proteins.     

Activation of NADPH oxidase 1 increases intracellular calcium and migration of smooth muscle cells

Redox-dependent migration and proliferation of vascular smooth muscle cells (SMCs) are central events in the development of vascular proliferative diseases; however, the underlying intracellular signaling mechanisms are not fully understood. We tested the hypothesis that activation of Nox1 NADPH oxidase modulates intracellular calcium ([Ca(2+)](i)) levels. Using cultured SMCs from wild-type and Nox1 null mice, we confirmed that thrombin-dependent generation of reactive oxygen species requires Nox1. Thrombin rapidly increased [Ca(2+)](i), as measured by fura-2 fluorescence ratio imaging, in wild-type but not Nox1 null SMCs. The increase in [Ca(2+)](i) in wild-type SMCs was inhibited by antisense to Nox1 and restored by expression of Nox1 in Nox1 null SMCs. Investigation into potential mechanisms by which Nox1 modulates [Ca(2+)](i) showed that thrombin-induced inositol triphosphate generation and thapsigargin-induced intracellular calcium mobilization were similar in wild-type and Nox1 null SMCs. To examine the effects of Nox1 on Ca(2+) entry, cells were either bathed in Ca(2+)-free medium or exposed to dihydropyridines to block L-type Ca(2+) channel activity. Treatment with nifedipine or removal of extracellular Ca(2+) reduced the thrombin-mediated increase of [Ca(2+)](i) in wild-type SMCs, whereas the response in Nox1 null SMCs was unchanged. Sodium vanadate, an inhibitor of protein tyrosine phosphatases, restored the thrombin-induced increase of [Ca(2+)](i) in Nox1 null SMCs. Migration of SMCs was impaired with deficiency of Nox1 and restored with expression of Nox1 or the addition of sodium vanadate. In summary, we conclude that Nox1 NADPH oxidase modulates Ca(2+) mobilization in SMCs, in part through regulation of Ca(2+) influx, to thereby promote cell migration.     

Is hyperlipidemia correlated with longer survival in patients with amyotrophic lateral sclerosis?

Amyotrophic lateral sclerosis (ALS) is the most serious form of degenerative motor neuron disease in adults, whose relentless course leads to death within 2-5 years, generally due to respiratory failure. Apart from the age and site of onset, no other factors have consistently demonstrated to be related to the ALS outcome. The aim of the study was to investigate the influence of fasting serum lipid levels (cholesterol and triglycerides) and the body mass index (BMI) at the time of diagnosis on survival in ALS patients. The study included 82 patients with ALS residing in the Belgrade area who were diagnosed with ALS over a time period of 4 years (2006-2009). Survival was assessed by the Kaplan-Meier method. In this retrospective study, 39 (47.56%) patients had normal values of lipids and 43 (52.43%) patients had hyperlipidemia. The mean survival time from the onset of symptoms for patients with normal lipidemia was 4.21 ± 0.5 years, while the mean survival time from the onset of symptoms for patients with hyperlipidemia was 5.0 ± 0.67 years (P = 0.36). We also did not register a significant difference in survival in relation to gender, the site or age of onset, even though we noticed a longer survival in patients with hyperlipidemia in all of the examined groups, especially in the group of younger patients, with the onset of the disease before the age of 45 years. If we take into account the fact that BMI is pathophysiologically associated with cholesterol and triglyceride serum levels, the results in our study complement each other showing that patients with a higher BMI, registered in 28.8% of the cases, do not live longer. Our findings show that hyperlipidemia, which we found in 52.43% of our ALS patients, at the time of diagnosis, is not related to significantly longer survival.     

Age-dependent effects of carotid endarterectomy or stenting on cognitive performance

Although evidence is accumulating that age modifies the risk of carotid angioplasty and stenting (CAS) versus endarterectomy (CEA) for patients with significant carotid stenosis, the impact of age on cognition after either CEA or CAS remains unclear. In this study, we analyzed the effects of age on cognitive performance after either CEA or CAS using a comprehensive neuropsychological test battery with parallel test forms and a control group to exclude a learning effect. The neuropsychological outcomes after revascularization were determined in 19 CAS and 27 CEA patients with severe carotid stenosis. The patients were subdivided according to their median age (<68?years and ≥68?years); 27 healthy subjects served as a control group. In all patients clinical examinations, MRI scans and a neuropsychological test battery that assessed four major cognitive domains were performed immediately before, within 72?h, and 3?months after CEA or CAS. While patients <68?years of age showed no significant cognitive alteration after either CEA or CAS, a significant cognitive decline was observed in patients ≥68?years in both treatment groups (p?=?0.001). Notably, this cognitive deterioration persisted in patients after CEA, whereas it was only transient in patients treated with CAS. These results demonstrate an age-dependent effect of CEA and CAS on cognitive functions. In contrast to the recently observed increased clinical complication rates in older subjects after CAS compared with CEA, CEA appears to be associated with a greater, persistent decline in cognitive performance than CAS in this subgroup of patients.