Tumor-Associated Macrophages in Glioblastoma Multiforme—A Suitable Target for Somatostatin Receptor-Based Imaging and Therapy?

Background

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. Tumor-associated macrophages (TAM) have been shown to promote malignant growth and to correlate with poor prognosis. [1,4,7,10-tetraazacyclododecane-NN′,N″,N′″-tetraacetic acid]-d-Phe1,Tyr3-octreotate (DOTATATE) labeled with Gallium-68 selectively binds to somatostatin receptor 2A (SSTR2A) which is specifically expressed and up-regulated in activated macrophages. On the other hand, the role of SSTR2A expression on the cell surface of glioma cells has not been fully elucidated yet. The aim of this study was to non-invasively assess SSTR2A expression of both glioma cells as well as macrophages in GBM.

Methods

15 samples of patient-derived GBM were stained immunohistochemically for macrophage infiltration (CD68), proliferative activity (Ki67) as well as expression of SSTR2A. Anti-CD45 staining was performed to distinguish between resident microglia and tumor-infiltrating macrophages. In a subcohort, positron emission tomography (PET) imaging using ⁶⁸Ga-DOTATATE was performed and the semiquantitatively evaluated tracer uptake was compared to the results of immunohistochemistry.

Results

The amount of microglia/macrophages ranged from <10% to >50% in the tumor samples with the vast majority being resident microglial cells. A strong SSTR2A immunostaining was observed in endothelial cells of proliferating vessels, in neurons and neuropile. Only faint immunostaining was identified on isolated microglial and tumor cells. Somatostatin receptor imaging revealed areas of increased tracer accumulation in every patient. However, retention of the tracer did not correlate with immunohistochemical staining patterns.

Conclusion

SSTR2A seems not to be overexpressed in GBM samples tested, neither on the cell surface of resident microglia or infiltrating macrophages, nor on the surface of tumor cells. These data suggest that somatostatin receptor directed imaging and treatment strategies are less promising in GBM.     

Somatostatin receptor subtype 4 modulates L-type calcium channels via Gβγ and PKC signaling in rat retinal ganglion cells

Somatostatin subtype-4 receptors (sst₄) inhibit L-type calcium channel currents (I_Ca) in retinal ganglion cells (RGCs). Here we identify the signaling pathways involved in sst₄ stimulation leading to suppression of I_Ca in RGCs. Whole cell patch clamp recordings were made on isolated immunopanned RGCs using barium as a charge carrier to isolate I_Ca. Application of the selective sst₄ agonist, L-803 (10 nM), reduced I_Ca by 41.2%. Pretreatment of cells with pertussis toxin (Gi/o inhibitor) did not prevent the action of L-803, which reduced I_Ca by 34.7%. To determine the involvement of Gβγ subunits after sst₄ activation, depolarizing pre-pulse facilitation paradigms were used to remove voltage-dependent inhibition of calcium channels. Pre-pulse facilitation did not reverse the inhibitory effects of L-803 on I_Ca (8.4 vs. 8.8% reductions, ctrl vs. L-803); however, pharmacologic inhibition of Gβγ reduced I_Ca suppression by L-803 (23.0%, P < 0.05). Inhibition of PKC (GF109203X; GFX) showed a concentration-dependent effect in preventing the action of L-803 on I_Ca (1 μM GFX, 34.3%; 5 μM GFX, 14.6%, P < 0.05). When both PKC and Gβγ were inhibited, the effects of L-803 on I_Ca were blocked (1.8%, P < 0.05). These results suggest that sst₄ stimulation modulates RGC calcium channels via Gβγ and PKC activation. Since reducing intracellular Ca²⁺ is known to be neuroprotective in RGCs, modulating these sst₄ signaling pathways may provide insights to the discovery of unique therapeutic targets to reduce intracellular Ca²⁺ levels in RGCs.     

Somatostatin receptor subtype 4 modulates L-type calcium channels via Gβγ and PKC signaling in rat retinal ganglion cells

Somatostatin subtype-4 receptors (sst₄) inhibit L-type calcium channel currents (I_Ca) in retinal ganglion cells (RGCs). Here we identify the signaling pathways involved in sst₄ stimulation leading to suppression of I_Ca in RGCs. Whole cell patch clamp recordings were made on isolated immunopanned RGCs using barium as a charge carrier to isolate I_Ca. Application of the selective sst₄ agonist, L-803 (10 nM), reduced I_Ca by 41.2%. Pretreatment of cells with pertussis toxin (Gi/o inhibitor) did not prevent the action of L-803, which reduced I_Ca by 34.7%. To determine the involvement of Gβγ subunits after sst₄ activation, depolarizing pre-pulse facilitation paradigms were used to remove voltage-dependent inhibition of calcium channels. Pre-pulse facilitation did not reverse the inhibitory effects of L-803 on I_Ca (8.4 vs. 8.8% reductions, ctrl vs. L-803); however, pharmacologic inhibition of Gβγ reduced I_Ca suppression by L-803 (23.0%, P < 0.05). Inhibition of PKC (GF109203X; GFX) showed a concentration-dependent effect in preventing the action of L-803 on I_Ca (1 μM GFX, 34.3%; 5 μM GFX, 14.6%, P < 0.05). When both PKC and Gβγ were inhibited, the effects of L-803 on I_Ca were blocked (1.8%, P < 0.05). These results suggest that sst₄ stimulation modulates RGC calcium channels via Gβγ and PKC activation. Since reducing intracellular Ca²⁺ is known to be neuroprotective in RGCs, modulating these sst₄ signaling pathways may provide insights to the discovery of unique therapeutic targets to reduce intracellular Ca²⁺ levels in RGCs.     

Somatostatin Derivate (smsDX) Attenuates the TAM-Stimulated Proliferation,Migration and Invasion of Prostate Cancer via NF-κB Regulation

Tumor development and progression are influenced by macrophages of the surrounding microenvironment. To investigate the influences of an inflammatory tumor microenvironment on the growth and metastasis of prostate cancer, the present study used a co-culture model of prostate cancer (PCa) cells with tumor-associated macrophage (TAM)-conditioned medium (MCM). MCM promoted PCa cell (LNCaP, DU145 and PC-3) growth, and a xenograft model in nude mice consistently demonstrated that MCM could promote tumor growth. MCM also stimulated migration and invasion in vitro. Somatostatin derivate (smsDX) significantly attenuated the TAM-stimulated proliferation, migration and invasion of prostate cancer. Immunohistochemistry revealed that NF-κB was over-expressed in PCa and BPH with chronic inflammatory tissue specimens and was positively correlated with macrophage infiltration. Further investigation into the underlying mechanism revealed that NF-κB played an important role in macrophage infiltration. SmsDX inhibited the paracrine loop between TAM and PCa cells and may represent a potential therapeutic agent for PCa.     

Recruitment of β-Arrestin into Neuronal Cilia Modulates Somatostatin Receptor Subtype 3 Ciliary Localization

Primary cilia are essential sensory and signaling organelles present on nearly every mammalian cell type. Defects in primary cilia underlie a class of human diseases collectively termed ciliopathies. Primary cilia are restricted subcellular compartments, and specialized mechanisms coordinate the localization of proteins to cilia. Moreover, trafficking of proteins into and out of cilia is required for proper ciliary function, and this process is disrupted in ciliopathies. The somatostatin receptor subtype 3 (Sstr3) is selectively targeted to primary cilia on neurons in the mammalian brain and is implicated in learning and memory. Here, we show that Sstr3 localization to cilia is dynamic and decreases in response to somatostatin treatment. We further show that somatostatin treatment stimulates β-arrestin recruitment into Sstr3-positive cilia and this recruitment can be blocked by mutations in Sstr3 that impact agonist binding or phosphorylation. Importantly, somatostatin treatment fails to decrease Sstr3 ciliary localization in neurons lacking β-arrestin 2. Together, our results implicate β-arrestin in the modulation of Sstr3 ciliary localization and further suggest a role for β-arrestin in the mediation of Sstr3 ciliary signaling.     

Octapeptide Analogs of Somatostatin Containing α,α-Dialkylated Amino Acids with Potent Anticancer Activity

We describe the synthesis and anticancer activities of octapeptide analogs of somatostatin incorporating α,α-dialkylated amino acids. The designed analogs of somatostatin are: d-Phe¹-Cys²-Tyr³-d-Trp⁴-Orn⁵-Xxx⁶-Pen⁷-Thr⁸-NH₂ where Xxx=α-Aminoisobutyric acid (Aib), Diethyl glycine (Deg), 1-Aminocyclopentane carboxylic acid (Ac5c), and, d-Phe¹-Cys²-Tyr³-d-Trp⁴-Lys⁵-Ac5c⁶-Pen⁷-Thr⁸-NH₂ (disulphide bond between Cys² and Pen⁷ in all analogs). The conformational studies two of the designed analogs were carried out by NMR techniques and the experimental results suggest a β-turn structure for one of the designed analog. In vivo tumor regression study of two designed analogs on human primary colon tumor xenografts in nude mice demonstrates the anticancer potential of the synthesized analogs.     

Somatostatin receptor-2 negatively regulates β-adrenergic receptor mediated Ca dependent signaling pathways in H9c2 cells

In the present study, we report that somatostatin receptor 2 (SSTR2) plays a crucial role in modulation of β₁AR and β₂AR mediated signaling pathways that are associated with increased intracellular Ca^2 + and cardiac complications. In H9c2 cells, SSTR2 colocalizes with β₁AR or β₂AR in receptor specific manner. SSTR2 selective agonist inhibits isoproterenol and formoterol stimulated cAMP formation and PKA phosphorylation in concentration dependent manner. In the presence of SSTR2 agonist, the expression of PKCα and PKCβ was comparable to the basal condition, however SSTR2 agonist inhibits isoproterenol or formoterol induced PKCα and PKCβ expression, respectively. Furthermore, the activation of SSTR2 not only inhibits calcineurin expression and its activity, but also blocks NFAT dephosphorylation and its nuclear translocation. SSTR2 selective agonist abrogates isoproterenol mediated increase in cell size and protein content (an index of hypertrophy). Taken together, the results described here provide direct evidence in support of cardiac protective role of SSTR2 via modulation of Ca^2 + associated signaling pathways attributed to cardiac hypertrophy.     

Syntheses and spectroscopic properties of α- and β-phosphatidylcholines and phosphatidylethanolamines

The widely used partial synthesis of phospholipids via deacylation of naturally occurring phospholipids, followed by reacylation with fatty acid anhydrides, is accompanied by phosphoryl migration. The resulting mixture of α- and β-phospholipids was separated by short-column chromatography. Milder acylation procedures in which no phosphoryl migration occurs, were developed. 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine was prepared in 50% yield by acylation of sn-glycero-3-phosphocholine (GPC) with N-linoleoylimidazole. Detailed NMR and infrared spectra of α- and β-phosphatidylcholines (PCs) and -ethanolamines (PEs) are reported and the differences between isomers discussed.     

Phenotypic plasticity and longevity in plants and animals: cause and effect?

Immobile plants and immobile modular animals outlive unitary animals. This paper discusses competing but not necessarily mutually exclusive theories to explain this extreme longevity, especially from the perspective of phenotypic plasticity. Stem cell immortality, vascular autonomy, and epicormic branching are some important features of the phenotypic plasticity of plants that contribute to their longevity. Monocarpy versus polycarpy can also influence the kind of senescent processes experienced by plants. How density-dependent phenomena affecting the establishment of juveniles in these immobile organisms can influence the evolution of senescence, and consequently longevity, is reviewed and discussed. Whether climate change scenarios will favour long-lived or short-lived organisms, with their attendant levels of plasticity, is also presented.     

Dogmatisms and Catechisms — Ethics and Companion Animals

Abstract

The current reconsideration of the place in nature of human beings unfortunately continues to be an acrimonious one. All too often the debate is more akin to a warlike encounter where each side attempts to gain control or the upper hand than a search for points of agreement. Given this context, it is important to entertain views that emanate from different cultural traditions as a way to infuse the debate with new life. Students of Native American culture have consistently pointed out that the essential concepts of life balance and reciprocity represented there may serve as useful points of consideration as we struggle with the appropriate relationships with animals and nature. This article presents a representative Zuni story, told by Governor Robert E. Lewis, that illustrates these notions.     

DCCP and DICP： Construction and Analyses of Databases for Copper- and Iron-Chelating Proteins

Copper and iron play important roles in a variety of biological processes, especially when being chelated with proteins. The proteins involved in the metal binding, transporting and metabolism have aroused much interest. To facilitate the study on this topic, we constructed two databases （DCCP and DICP） containing the known copper- and iron-chelating proteins~ which are freely available from the website http：//sdbi.sdut.edu.cn/en. Users can conveniently search and browse all of the entries in the databases. Based on the two databases, bioinformatic analyses were performed, which provided some novel insights into metalloproteins.     

Biosynthesis of geraniol and nerol and their β-d-glucosides in Perlargonium graveolens and Rosa dilecta

1. 3R-[2-(14)C]Mevalonate was incorporated into geranyl and neryl beta-d-glucosides in petals of Rosa dilecta in up to 10.6% yield, and the terpenoid part was specifically and equivalently labelled in the moieties derived from isopentenyl pyrophosphate and 3,3-dimethylallyl pyrophosphate. A similar labelling pattern, with incorporations of 0.06-0.1% was found for geraniol or nerol formed in leaves of Pelargonium graveolens The former results provide the best available evidence for the mevalonoid route to regular monoterpenes in higher plants. 2. Incorporation studies with 3RS-[2-(14)C,(4R)-4-(3)H(1)]-mevalonate and its (4S)-isomer showed that the pro-4R hydrogen atom of the precursor was retained and the pro-4S hydrogen atom was eliminated in both alcohols and both glucosides. These results suggest that the correlation of retention of the pro-4S hydrogen atom of mevalonate with formation of a cis-substituted double bond, such as has been found in certain higher terpenoids, does not apply to the biosynthesis of monoterpenes. It is proposed that either nerol is derived from isomerization of geraniol or the two alcohols are directly formed by different prenyltransferases. Possible mechanisms for these processes are discussed. 3. The experiments with [(14)C,(3)H]mevalonate also show that in these higher plants, as has been previously found in animal tissue and yeast, the pro-4S hydrogen atom of mevalonate was lost in the conversion of isopentenyl pyrophosphate into 3,3-dimethylallyl pyrophosphate.     

Zinc and diabetes — clinical links and molecular mechanisms

Zinc is an essential trace element crucial for the function of more than 300 enzymes and it is important for cellular processes like cell division and apoptosis. Hence, the concentration of zinc in the human body is tightly regulated and disturbances of zinc homeostasis have been associated with several diseases including diabetes mellitus, a disease characterized by high blood glucose concentrations as a consequence of decreased secretion or action of insulin. Zinc supplementation of animals and humans has been shown to ameliorate glycemic control in type 1 and 2 diabetes, the two major forms of diabetes mellitus, but the underlying molecular mechanisms have only slowly been elucidated. Zinc seems to exert insulin-like effects by supporting the signal transduction of insulin and by reducing the production of cytokines, which lead to beta-cell death during the inflammatory process in the pancreas in the course of the disease. Furthermore, zinc might play a role in the development of diabetes, since genetic polymorphisms in the gene of zinc transporter 8 and in metallothionein (MT)-encoding genes could be demonstrated to be associated with type 2 diabetes mellitus. The fact that antibodies against this zinc transporter have been detected in type 1 diabetic patients offers new diagnostic possibilities. This article reviews the influence of zinc on the diabetic state including the molecular mechanisms, the role of the zinc transporter 8 and MT for diabetes development and the resulting diagnostic and therapeutic options.     

siRNA, miRNA and HIV： promises and challenges

Small interfering RNA （siRNA） and microRNA （miRNA） are small RNAs of 18-25 nucleotides （nt） in length that play important roles in regulating gene expression. They are incorporated into an RNA-induced silencing complex （RISC） and serve as guides for silencing their corresponding target mRNAs based on complementary base-pairing. The promise of gene silencing has led many researchers to consider siRNA as an anti-viral tool. However, in long-term settings, many viruses appear to escape from this therapeutical strategy. An example of this may be seen in the case of human immunodeficiency virus type-1 （HIV-1） which is able to evade RNA silencing by either mutating the siRNA-targeted sequence or by encoding for a partial suppressor of RNAi （RNA interference）. On the other hand, because miRNA targeting does not require absolute complementarity of base-pairing, mutational escape by viruses from miRNA-specified silencing may be more difficult to achieve. In this review, we discuss stratagems used by various viruses to avoid the cells＇ antiviral si/mi-RNA defenses and notions of how viruses might control and regulate host cell genes by encoding viral miRNAs （vmiRNAs）.     

Toxoplasmosis and Epilepsy — Systematic Review and Meta Analysis

Background

Toxoplasmosis is an important, widespread, parasitic infection caused by Toxoplasma gondii. The chronic infection in immunocompetent patients, usually considered as asymptomatic, is now suspected to be a risk factor for various neurological disorders, including epilepsy. We aimed to conduct a systematic review and meta-analysis of the available literature to estimate the risk of epilepsy due to toxoplasmosis.

Methods

A systematic literature search was conducted of several databases and journals to identify studies published in English or French, without date restriction, which looked at toxoplasmosis (as exposure) and epilepsy (as disease) and met certain other inclusion criteria. The search was based on keywords and suitable combinations in English and French. Fixed and random effects models were used to determine odds ratios, and statistical significance was set at 5.0%.

Principal findings

Six studies were identified, with an estimated total of 2888 subjects, of whom 1280 had epilepsy (477 positive for toxoplasmosis) and 1608 did not (503 positive for toxoplasmosis). The common odds ratio (calculated) by random effects model was 2.25 (95% CI 1.27–3.9), p = 0.005.

Conclusions

Despite the limited number of studies, and a lack of high-quality data, toxoplasmosis should continue to be regarded as an epilepsy risk factor. More and better studies are needed to determine the real impact of this parasite on the occurrence of epilepsy.     

Rise and Demise of Bioinformatics? Promise and Progress

The field of bioinformatics and computational biology has gone through a number of transformations during the past 15 years, establishing itself as a key component of new biology. This spectacular growth has been challenged by a number of disruptive changes in science and technology. Despite the apparent fatigue of the linguistic use of the term itself, bioinformatics has grown perhaps to a point beyond recognition. We explore both historical aspects and future trends and argue that as the field expands, key questions remain unanswered and acquire new meaning while at the same time the range of applications is widening to cover an ever increasing number of biological disciplines. These trends appear to be pointing to a redefinition of certain objectives, milestones, and possibly the field itself.