Cytokine Plasma Levels: Reliable Predictors for Radiation Pneumonitis?

Background

Radiotherapy (RT) is the primary treatment modality for inoperable, locally advanced non-small-cell lung cancer (NSCLC), but even with highly conformal treatment planning, radiation pneumonitis (RP) remains the most serious, dose-limiting complication. Previous clinical reports proposed that cytokine plasma levels measured during RT allow to estimate the individual risk of patients to develop RP. The identification of such cytokine risk profiles would facilitate tailoring radiotherapy to maximize treatment efficacy and to minimize radiation toxicity. However, cytokines are produced not only in normal lung tissue after irradiation, but are also over-expressed in tumour cells of NSCLC specimens. This tumour-derived cytokine production may influence circulating plasma levels in NSCLC patients. The aim of the present study was to investigate the prognostic value of TNF-α, IL-1β, IL-6 and TGF-β1 plasma levels to predict radiation pneumonitis and to evaluate the impact of tumour-derived cytokine production on circulating plasma levels in patients irradiated for NSCLC.

Methodology/Principal Findings

In 52 NSCLC patients (stage I–III) cytokine plasma levels were investigated by ELISA before and weekly during RT, during follow-up (1/3/6/9 months after RT), and at the onset of RP. Tumour biopsies were immunohistochemically stained for IL-6 and TGF-β1, and immunoreactivity was quantified (grade 1–4). RP was evaluated according to LENT-SOMA scale. Tumour response was assessed according to RECIST criteria by chest-CT during follow-up. In our clinical study 21 out of 52 patients developed RP (grade I/II/III/IV: 11/3/6/1 patients). Unexpectedly, cytokine plasma levels measured before and during RT did not correlate with RP incidence. In most patients IL-6 and TGF-β1 plasma levels were already elevated before RT and correlated significantly with the IL-6 and TGF-β1 production in corresponding tumour biopsies. Moreover, IL-6 and TGF-β1 plasma levels measured during follow-up were significantly associated with the individual tumour responses of these patients.

Conclusions/Significance

The results of this study did not confirm that cytokine plasma levels, neither their absolute nor any relative values, may identify patients at risk for RP. In contrast, the clear correlations of IL-6 and TGF-β1 plasma levels with the cytokine production in corresponding tumour biopsies and with the individual tumour responses suggest that the tumour is the major source of circulating cytokines in patients receiving RT for advanced NSCLC.     

rac regulates its effector phospholipase Cgamma2 through interaction with a split pleckstrin homology domain

Several isoforms of phospholipase C (PLC) are regulated through interactions with Ras superfamily GTPases, including Rac proteins. Interestingly, of two closely related PLCgamma isoforms, only PLCgamma(2) has previously been shown to be activated by Rac. Here, we explore the molecular basis of this interaction as well as the structural properties of PLCgamma(2) required for activation. Based on reconstitution experiments with isolated PLCgamma variants and Rac2, we show that an unusual pleckstrin homology (PH) domain, designated as the split PH domain (spPH), is both necessary and sufficient to effect activation of PLCgamma(2) by Rac2. We also demonstrate that Rac2 directly binds to PLCgamma(2) as well as to the isolated spPH of this isoform. Furthermore, through the use of NMR spectroscopy and mutational analysis, we determine the structure of spPH, define the structural features of spPH required for Rac interaction, and identify critical amino acid residues at the interaction interface. We further discuss parallels and differences between PLCgamma(1) and PLCgamma(2) and the implications of our findings for their respective signaling roles.     

Chloride Is essential for capacitation and for the capacitation-associated increase in tyrosine phosphorylation

After epididymal maturation, sperm capacitation, which encompasses a complex series of molecular events, endows the sperm with the ability to fertilize an egg. This process can be mimicked in vitro in defined media, the composition of which is based on the electrolyte concentration of the oviductal fluid. It is well established that capacitation requires Na(+), HCO(3)(-), Ca(2+), and a cholesterol acceptor; however, little is known about the function of Cl(-) during this important process. To determine whether Cl(-), in addition to maintaining osmolarity, actively participates in signaling pathways that regulate capacitation, Cl(-) was replaced by either methanesulfonate or gluconate two nonpermeable anions. The absence of Cl(-) did not affect sperm viability, but capacitation-associated processes such as the increase in tyrosine phosphorylation, the increase in cAMP levels, hyperactivation, the zona pellucidae-induced acrosome reaction, and most importantly, fertilization were abolished or significantly reduced. Interestingly, the addition of cyclic AMP agonists to sperm incubated in Cl(-)-free medium rescued the increase in tyrosine phosphorylation and hyperactivation suggesting that Cl(-) acts upstream of the cAMP/protein kinase A signaling pathway. To investigate Cl(-) transport, sperm incubated in complete capacitation medium were exposed to a battery of anion transport inhibitors. Among them, bumetanide and furosemide, two blockers of Na(+)/K(+)/Cl(-) cotransporters (NKCC), inhibited all capacitation-associated events, suggesting that these transporters may mediate Cl(-) movements in sperm. Consistent with these results, Western blots using anti-NKCC1 antibodies showed the presence of this cotransporter in mature sperm.     

G-protein-coupled receptor-mediated traffic of Na,K-ATPase to the plasma membrane requires the binding of adaptor protein 1 to a Tyr-255-based sequence in the alpha-subunit

Motion of integral membrane proteins to the plasma membrane in response to G-protein-coupled receptor signals requires selective cargo recognition motifs that bind adaptor protein 1 and clathrin. Angiotensin II, through the activation of AT1 receptors, promotes the recruitment to the plasma membrane of Na,K-ATPase molecules from intracellular compartments. We present evidence to demonstrate that a tyrosine-based sequence (IVVY-255) present within the Na,K-ATPase alpha1-subunit is involved in the binding of adaptor protein 1. Mutation of Tyr-255 to a phenylalanine residue in the Na,K-ATPase alpha1-subunit greatly reduces the angiotensin II-dependent activation of Na,K-ATPase, recruitment of Na,K-ATPase molecules to the plasma membrane, and association of adaptor protein 1 with Na,K-ATPase alpha1-subunit molecules. To determine protein-protein interaction, we used fluorescence resonance energy transfer between fluorophores attached to the Na,K-ATPase alpha1-subunit and adaptor protein 1. Although angiotensin II activation of AT1 receptors induces a significant increase in the level of fluorescence resonance energy transfer between the two molecules, this effect was blunted in cells expressing the Tyr-255 mutant. Thus, results from different methods and techniques suggest that the Tyr-255-based sequence within the NKA alpha1-subunit is the site of adaptor protein 1 binding in response to the G-protein-coupled receptor signals produced by angiotensin II binding to AT1 receptors.     

What do we know about serotonin?

The present review focuses on what is known of basic serotonin physiology in the human body. Here, we describe serotonin biochemistry and metabolism and summarize the results of studies that have contributed significantly to our understanding of serotonin physiology. We report the well-established role of serotonin in cardiovascular, gastrointestinal, and circulatory physiology. Emphasis is placed on the role of serotonin in peripheral physiological systems rather than in the central nervous system. A brief overview is provided on the emerging role of serotonin in novel areas such as bone pathways and glucose uptake. We also report a select few animal studies and animal models that have provided worthwhile contributions to the understanding of serotonin in human physiology. In addition, we summarize the results of large-scale genetic studies on serotonin and serotonin transporter genes, performed in relation to behavioral and mood disorders.     

Structural basis for a distinct catalytic mechanism in Trypanosoma brucei tryparedoxin peroxidase

Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three cysteine homologues (Px I-III) of classical selenocysteine-containing glutathione peroxidases. The enzymes obtain their reducing equivalents from the unique trypanothione (bis(glutathionyl)spermidine)/tryparedoxin system. During catalysis, these tryparedoxin peroxidases cycle between an oxidized form with an intramolecular disulfide bond between Cys(47) and Cys(95) and the reduced peroxidase with both residues in the thiol state. Here we report on the three-dimensional structures of oxidized T. brucei Px III at 1.4A resolution obtained by x-ray crystallography and of both the oxidized and the reduced protein determined by NMR spectroscopy. Px III is a monomeric protein unlike the homologous poplar thioredoxin peroxidase (TxP). The structures of oxidized and reduced Px III are essentially identical in contrast to what was recently found for TxP. In Px III, Cys(47), Gln(82), and Trp(137) do not form the catalytic triad observed in the selenoenzymes, and related proteins and the latter two residues are unaffected by the redox state of the protein. The mutational analysis of three conserved lysine residues in the vicinity of the catalytic cysteines revealed that exchange of Lys(107) against glutamate abrogates the reduction of hydrogen peroxide, whereas Lys(97) and Lys(99) play a crucial role in the interaction with tryparedoxin.     

Uptake and recycling of pro-BDNF for transmitter-induced secretion by cortical astrocytes

Activity-dependent secretion of brain-derived neurotrophic factor (BDNF) is thought to enhance synaptic plasticity, but the mechanisms controlling extracellular availability and clearance of secreted BDNF are poorly understood. We show that BDNF is secreted in its precursor form (pro-BDNF) and is then cleared from the extracellular space through rapid uptake by nearby astrocytes after θ-burst stimulation in layer II/III of cortical slices, a paradigm resulting in long-term potentiation of synaptic transmission. Internalization of pro-BDNF occurs via the formation of a complex with the pan-neurotrophin receptor p75 and subsequent clathrin-dependent endocytosis. Fluorescence-tagged pro-BDNF and real-time total internal reflection fluorescence microscopy in cultured astrocytes is used to monitor single endocytic vesicles in response to the neurotransmitter glutamate. We find that endocytosed pro-BDNF is routed into a fast recycling pathway for subsequent soluble NSF attachment protein receptor–dependent secretion. Thus, astrocytes contain an endocytic compartment competent for pro-BDNF recycling, suggesting a specialized form of bidirectional communication between neurons and glia.     

AVPR2 variants and mutations in nephrogenic diabetes insipidus: review and missense mutation significance

Almost 90% of nephrogenic diabetes insipidus (NDI) is due to mutations in the arginine-vasopressin receptor 2 gene (AVPR2). We retrospectively examined all the published mutations/variants in AVPR2. We planned to perform a comprehensive review of all the AVPR2 mutations/variants and to test whether any amino acid change causing a missense mutation is significantly more or less common than others. We performed a Medline search and collected detailed information regarding all AVPR2 mutations and variants. We performed a frequency comparison between mutated and wild-type amino acids and codons. We predicted the mutation effect or reported it based on published in vitro studies. We also reported the ethnicity of each mutation/variant carrier. In summary, we identified 211 AVPR2 mutations which cause NDI in 326 families and 21 variants which do not cause NDI in 71 NDI families. We described 15 different types of mutations including missense, frameshift, inframe deletion, deletion, insertion, nonsense, duplication, splicing and combined mutations. The missense mutations represent the 55.83% of all the NDI published families. Arginine and tyrosine are significantly (P = 4.07E-08 and P = 3.27E-04, respectively) the AVPR2 most commonly mutated amino acids. Alanine and glutamate are significantly (P = 0.009 and P = 0.019, respectively) the least mutated AVPR2 amino acids. The spectrum of mutations varies from rare gene variants or polymorphisms not causing NDI to rare mutations causing NDI, among which arginine and tyrosine are the most common missense. The AVPR2 mutations are spread world-wide. Our study may serve as an updated review, comprehensive of all AVPR2 variants and specific gene locations. J. Cell. Physiol. 217: 605-617, 2008. (c) 2008 Wiley-Liss, Inc.     

Inflammatory cytokines increase extracellular procathepsin D in permanent and primary endothelial cell cultures

The protease cathepsin D (Cath D) and its proteolytically inactive proform, procathepsin D (ProCath D), turned out to be multifunctional within and outside the cell. Elevated levels of ProCath D occur in malignant tumors and in organs under chronic inflammation. One important source for this increase of ProCath D might be endothelial cells. Here we examined the expression of Cath D in the human endothelial cell line EA.hy 926 and in primary endothelial cells isolated from human umbilical cord veins (HUVEC). After serum-free incubation with or without human interferon-gamma (hIFN-gamma) and/or human tumor necrosis factor-alpha (hTNF-alpha) immature and mature Cath D forms were examined in cell extracts and in cell-conditioned medium concentrates by Western blotting. Lysates of EA.hy 926 cells as well as of HUVEC contained active Cath D as two-chain form, but only negligible amounts of ProCath D and Cath D intermediates. Yet both endothelial cell cultures accumulated ProCath D in their conditioned media in the absence of any stimulus. The treatment with hIFN-gamma and/or hTNF-alpha had little effect on intracellular levels of Cath D, whereas the cytokine stimulation increased the extracellular presence of ProCath D in both endothelial cell cultures. The extracellular increase of ProCath D was not related to induction of apoptosis, as validated by cleaved caspase-3 in cell lysates. Acidification of cytokine-treated media converted ProCath D into Cath D, which was associated with cathepsin-like activity using a fluorogenic substrate-linked assay. We conclude, in vitro, endothelial cells are a cytokine-dependent source for extracellular ProCath D.