Metastasis-directed Therapy in Prostate Cancer: Prognostic Significance of the ESTRO/EORTC Classification in Oligometastatic Bone Disease.

AIMS: Oligometastatic disease (OMD) represents a spectrum of clinical scenarios and various classification systems have been proposed. Bone-only OMD can occur in patients with advanced prostate cancer and validated decision-making tools are needed to assist patient selection for metastasis-directed therapy. The aim of the present study was to determine the prognostic utility of a classification system for OMD. MATERIALS AND METHODS: A retrospective review was conducted of all patients with bone-only oligometastatic prostate cancer treated with stereotactic body radiotherapy (SBRT) since November 2011. SBRT was delivered using CyberKnife® and gantry-based linear accelerator platforms. All patients were classified into oligometastatic states based on the European Society for Radiotherapy and Oncology/European Organisation for Research and Treatment of Cancer (ESTRO/EORTC) classification system. Kaplan-Meier and Cox regression analyses were carried out to determine the prognostic utility of this classification system. RESULTS: In total, 105 patients with 145 osseous metastases were treated over 119 sessions. The median follow-up after SBRT was 23 months (interquartile range 10-39.8). Twelve patients had died after a median time of 31 months. The 3-year metastatic progression-free survival was 23% (95% confidence interval 13-32) and the 3-year overall survival was 88% (95% confidence interval 80-96). Patients in a metachronous oligometastatic state were 4.50 (95% confidence interval 1.19-17.10, P = 0.03) times more likely to experience metastatic progression compared with those with synchronous oligometastases, and 6.69 (95% confidence interval 1.05-42.50, P = 0.04) times more likely to experience any failure. Hazard ratio magnitudes increased for patients in a repeat oligometastatic state. The multivariate model for both metastatic progression-free survival and failure-free survival found prostate-specific antigen doubling time <4 months (P = 0.002; P = 0.05) to independently predict for progression. CONCLUSION: The ESTRO/EORTC classification of OMD predicts for progression in patients treated with SBRT for bone-only oligometastatic prostate cancer at our institution. Further validation in prospective series over multiple tumour sites is needed. These characterisation factors should be assessed in patients considered for metastasis-directed therapy together with established prognostic features.

Oxygen-dependent modulation of erythropoietin mRNA levels in isolated rat kidneys studied by RNase protection.

Using oligonucleotide primers complementary to conserved regions in the mouse erythropoietin (Epo) gene, a portion of the rat Epo gene was amplified by the polymerase chain reaction to produce a probe suitable for assay of rat Epo mRNA by RNAse protection. The assay, which has sufficient sensitivity to measure to Epo mRNA in unstimulated rat kidneys, was used to demonstrate high amplitude in vitro modulation of Epo mRNA levels in response to changes in perfusate flow rate and oxygen tension in isolated kidneys, thus providing clear evidence that all the necessary events linking changes in oxygen delivery to the modulation of Epo mRNA levels can occur intrarenally.

Radiological Kinetics of Brain Metastases and Clinical Implications for Patients Treated With Stereotactic Radiosurgery.

AIMS: Select patients with brain metastases receive stereotactic radiosurgery (SRS) with the objective of improving survival and intracranial disease control. Brain metastases number and volume are prognostic factors used to inform patient selection. The aim of this study was to assess the rate of change of brain metastases size and number (growth kinetics) between the diagnostic and day of SRS magnetic resonance imaging (MRI) scans. MATERIALS AND METHODS: All patients treated with Gamma Knife SRS between October 2015 and April 2017 were included in this single-centre retrospective analysis. Brain metastases number and diameter were recorded at diagnosis and treatment. For patients with multiple brain metastases, the largest lesion was the index lesion. Distant intracranial control and overall survival were reported from the date of SRS. RESULTS: In total, 146 patients received 156 episodes of SRS. The median interval between diagnostic and SRS MRI was 20 days (range 1-68). Interval growth in the index lesion of at least 3 mm or the development of a new brain metastasis was noted in 60.2% of patients. This was associated with age less than 60 years (P = 0.001), Eastern Cooperative Oncology Group (ECOG) performance status 2 or above (P = 0.04), non-small cell lung carcinoma (NSCLC) (P = 0.03) or melanoma histologies (P = 0.05) and uncontrolled extracranial disease (P = 0.05). These patients were also more likely to develop distant intracranial recurrence (P = 0.046). Clinically significant growth was not associated with scan interval or differences in overall survival. The Kaplan-Meier estimate of probability of survival at 12 months was 59.3% (95% confidence interval 46.7-75.2%) for all patients. CONCLUSION: Intracranial progression between diagnosis and day of SRS is common. Risk factors are uncontrolled extracranial disease, poorer performance status, NSCLC or melanoma histologies and age less than 60 years. These patients would benefit from an MRI closer to treatment to inform patient selection and target delineation for SRS planning.

Imaging Ca2+ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon.

Regulated exocytosis involves the Ca(2+)-triggered fusion of secretory vesicles with the plasma membrane, by activation of vesicle membrane Ca(2+)-binding proteins [1]. The Ca(2+)-binding sites of these proteins are likely to lie within 30 nm of the vesicle surface, a domain in which changes in Ca2+ concentration cannot be resolved by conventional fluorescence microscopy. A fluorescent indicator for Ca2+ called a yellow 'cameleon' (Ycam2) - comprising a fusion between a cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13 and an enhanced yellow-emitting GFP - which is targetable to specific intracellular locations, has been described [2]. Here, we generated a fusion between phogrin, a protein that is localised to secretory granule membranes [3], and Ycam2 (phogrin-Ycam2) to monitor changes in Ca2+ concentration ([Ca2+]) at the secretory vesicle surface ([Ca2+]gd) through alterations in fluorescence resonance energy transfer (FRET) between the linked cyan and yellow fluorescent proteins (CFP and YFP, respectively) in Ycam2. In both neuroendocrine PC12 and MIN6 pancreatic beta cells, apparent resting values of cytosolic [Ca2+] and [Ca2+](gd) were similar throughout the cell. In MIN6 cells following the activation of Ca2+ influx, the minority of vesicles that were within approximately 1 microm of the plasma membrane underwent increases in [Ca2+](gd) that were significantly greater than those experienced by deeper vesicles, and greater than the apparent cytosolic [Ca2+] change. The ability to image both global and compartmentalised [Ca2+] changes with recombinant targeted cameleons should extend the usefulness of these new Ca2+ probes.

Mechanism of ischemic mitral regurgitation with segmental left ventricular dysfunction: three-dimensional echocardiographic studies in models of acute and chronic progressive regurgitation.

OBJECTIVES: This study aimed to separate proposed mechanisms for segmental ischemic mitral regurgitation (MR), including left ventricular (LV) dysfunction versus geometric distortion by LV dilation, using models of acute and chronic segmental ischemic LV dysfunction evaluated by three-dimensional (3D) echocardiography. BACKGROUND: Dysfunction and dilation-both mechanisms with practical therapeutic implications-are difficult to separate in patients. METHODS: In seven dogs with acute left circumflex (LCX) coronary ligation, LV expansion was initially restricted and then permitted to occur. In seven sheep with LCX branch ligation, LV expansion was also initially limited but became prominent with remodeling over eight weeks. Three-dimensional echo reconstruction quantified mitral apparatus geometry and MR volume. RESULTS: In the acute model, despite LV dysfunction with ejection fraction = 23 +/- 8%, MR was initially trace with limited LV dilation, but it became moderate with subsequent prominent dilation. In the chronic model, MR was also initially trace, but it became moderate over eight weeks as the LV dilated and changed shape. In both models, the only independent predictor of MR volume was increased tethering distance from the papillary muscles (PMs) to the anterior annulus, especially medial and posterior shift of the ischemic medial PM, measured by 3D reconstruction (r2 = 0.75 and 0.86, respectively). Mitral regurgitation volume did not correlate with LV ejection fraction or dP/dt. CONCLUSIONS: Segmental ischemic LV contractile dysfunction without dilation, even in the PM territory, fails to produce important MR. The development of MR relates strongly to changes in the 3D geometry of the mitral apparatus, with implications for approaches to restore a more favorable configuration.

Effect of bacterial endotoxin on the in vitro release of Type II phospholipase-A2 and prostaglandin E2 from human placenta.

The aim of this study was to establish the effect of bacterial endotoxin lipopolysaccharide (LPS) on the release of Type II phospholipase-A2 (PLA2) and prostaglandin E2 (PGE2) from late-pregnant human placental tissue incubated in vitro. Under basal conditions, immunoreactive Type II PLA2 and PGE2 were released from tissue explants in a time-dependent manner (up to 24 h, ANOVA, P<0. 0001, n=6). The release of these mediators was not associated with a loss of cell membrane integrity, as indicated by concentrations of the intracellular enzyme, lactate dehydrogenase (LDH), in the incubation medium. Incubation of explants in the presence of LPS (0. 001-100 microg LPS/ml) significantly decreased PLA2 tissue content (P<0.02, n=6) and increased the accumulation of PLA2 and PGE2 in the incubation medium (P<0.0001, n=6). The data obtained in this study indicated that Type II PLA2 and PGE2 are released from term placenta under basal conditions and that LPS stimulated their release. The associated decrease in PLA2 tissue content is consistent with the hypothesis that LPS induces the release of stored PLA2. This study identifies one pathway by which products of bacterial infection may induce the release of a pro-inflammatory, extracellularly active PLA2 from intrauterine tissues that may promote the formation of phospholipid metabolites involved in the process of labour and delivery (e.g. the prostaglandins).

The effect of monophosphoryl lipid A on lipopolysaccharide-induced prostaglandin E2 release in human choriodecidua.

The aim of this study was to determine the effect of an inhibitor of bacterial endotoxin, monophosphoryl lipid A (MLA), on lipopolysaccharide (LPS)-induced prostaglandin E2 (PGE2) formation by human choriodecidua explants incubated in vitro. LPS induced the release of PGE2 from explants in a time-and dose-dependent manner (P < 0.05, n = 5), thus establishing the efficacy of the experimental model. MLA at concentrations of 10 micrograms/ml also increased PGE2 release from explants when compared to vehicle controls (P < 0.05, n = 5). When used at a concentration that did not stimulate PGE2 release (1 microgram/ml), MLA pretreatment, coincubation or a combination of these protocols did not significantly affect LPS-induced PGE2 release. These data establish that MLA does not act by abrogating tissue LPS responsiveness. Under the conditions utilized in this study, MLA acts locally as a low potency 'LPS-like agent'. The previously reported in vivo efficacy of systemically administered MLA may involve the partial depletion or down regulation of LPS response pathways and the subsequent development of LPS tolerance.

Effect of inhaled alpha-emitting nuclides on mouse alveolar macrophages.

The effects of inhaled alpha emitters on the free cell population of the mouse lung were investigated up to 100 days after exposure. Groups of mice inhaled aerosols of 238PuO2, 239PuO2, or 241Am(NO3)3 to give alveolar deposits resulting in lung-averaged cumulative absorbed doses of about 20 Gy by the end of the study. Initially, with 238Pu most of the activity was associated with relatively few pulmonary alveolar macrophages (PAM), whereas with 241Am, all pulmonary alveolar macrophages were labeled and a substantial fraction was extracellular. The free cell population of the lung was sampled using bronchoalveolar lavage. The main parameters investigated were (a) the recovery and total numbers of free cells, including PAM, lymphocytes, and neutrophils; (b) the incidence of nuclear abnormalities in PAM (cells with more than one nucleus or with micronuclei); and (c) metabolic activation of PAM from measurements of their size and associated beta-glucuronidase activity. All three actinides produced depletions in total numbers of PAM, increased incidences of nuclear abnormalities, and metabolic activation of PAM, without a marked infiltration of inflammatory cells. Americium-241, which is distributed relatively uniformly in PAM, produced the most marked changes in that population and 238Pu, which gave the most inhomogeneous distribution of activity, produced the least.

Controlled active motion following primary flexor tendon repair: a prospective study over 9 years.

One hundred and thirty patients with 339 divided flexor tendons affecting 208 fingers were studied prospectively between 1988 and 1996, to assess a regime of primary flexor tendon suture and active postoperative motion, combined with a modified Kleinert dynamic traction splint. The tendon suture technique used was a high-strength multistrand technique using a modified Kessler core and a Halsted peripheral stitch. The results were influenced by the zone in which the tendon was divided, by the physiotherapy and to a lesser extent by the grade of surgeon operating. Overall results by Strickland criteria were 92% excellent or good, 7% fair and 1% poor. There were 43 complications in 31 patients including five zone 2 ruptures (5.7%) and one further rupture in zone 5. This method of flexor tendon repair requires good physiotherapy and splint-making capability but gives good results with minimal need for further surgery.

Proton magnetic resonance study of conformational dynamics, coordianted internal motions, and chemical shifts of tocinamide.

All proton magnetic resonance lines of the NH-CHalpha-CH2beta fragments of five amino acid residues of the heterodetic peptide, tocinamide, have been analyzed, including reassigment of certain NH, CHalpha, and CH2beta resonances. The 1H NMR spectral parameters evaluated from this analysis include (a) all chemical shifts, (b) (3JNHCH), (3Jalpha beta), and (2Jbeta beta) values, and (c) temperature dependencies of these coupling constants and chemical shifts--the latter includes aliphatic and aromatic CH protons. The (3Jalpha beta) coupling constants yielded Calpha-Cbeta rotamer populations, Pi, for five residues and these were used to explore conformational dynamics and coordinated internal motions of tocinamide. The rotamer populations and their temperature dependencies established, (1) that all Calpha-Cbeta bonds exhibit extensive internal rotation; (2) that rotamer preferences exist for every residue; (3) that the 162 conformations possible for completely free rotation around every bond of the Calpha1-Cbeta1-S1-S6-Cbeta6-Calpha6 disulfide fragment can be reduced to two or three if the concept of coordinated internal motion is introduced. We reject the possibility of a frozen conformation for the Cys1-Cys6 fragment in the eclipsed, staggered, or nonclassical rotamer states and propose the following possibilities: (a) three classical rotamers populated in the ratio 60:25:15, (b) three nonclassical rotamers not differing by +/- 15 degrees from the classical values of 180 degrees, + 60 degrees, and -60 degrees, or (c) two nonclassical but noneclipsed rotamers with a third rotamer essentially zero, and (4) that the Calpha-Cbeta rotamer populations of the Tyr2-Cys1-S-S-Cys6 fragment are consistent with coordinated internal motions of the Calpha-Cbeta bonds of all three residues. The existence of temperature coefficients, deltadelta/deltaT, POF +/- 2 ppb/degrees C for aliphatic protons can be accounted for, in part, by postulating intrinsic rotamer chemical shifts which are temperature independent. Deltadelta/deltaT values should therefore be useful for investigating conformational dynamics. A further consequence of this interpretation is that greater care should be exercised in using deltadelta/deltaT values to assign hydrogen-bonded amide protons, especially when deltadelta/deltaT approximately -2 to -4 ppb/degrees C.

Identification of a novel genetic locus that is required for in vitro adhesion of a clinical isolate of enterohaemorrhagic Escherichia coli to epithelial cells.

Enterohaemorrhagic Escherichia coli (EHEC) are food-borne intestinal pathogens with a low infectious dose. Adhesion of some EHEC strains to epithelial cells is attributed, in part, to intimin, but other factors may be required for the intestinal colonizing ability of these bacteria. In order to identify additional adherence factors of EHEC, we generated transposon mutants of a clinical EHEC isolate of serotype O111:H-, which displayed high levels of adherence to cultured Chinese hamster ovary (CHO) cells. One mutant was markedly deficient in CHO cell adherence, human red blood cell agglutination and autoaggregation. Sequence analysis of the gene disrupted in this mutant revealed a 9669 bp novel chromosomal open reading frame (ORF), which was designated efa1, for EHEC factor for adherence. efa1 displayed 28% amino acid identity with the predicted product of a recently described ORF from the haemolysin-encoding plasmid of EHEC O157:H7. The amino termini of the putative products of these two genes exhibit up to 38% amino acid similarity to Clostridium difficile toxins A and B. efa1 occurred within a novel genetic locus, at least 15 kb in length, which featured a low G+C content, several insertion sequence homologues and a homologue of the Shigella flexneri enterotoxin ShET2. DNA probes prepared from different regions of efa1 hybridized with all of 116 strains of attaching-effacing E. coli (AEEC) of a variety of serotypes, including enteropathogenic E. coli (EPEC) and EHEC, but with none of 91 non-AEEC strains. Nevertheless, efa1 was not required for the attachment-effacement phenotype, and the efa1 locus was not physically linked to the locus for enterocyte effacement (LEE) pathogenicity island, which is responsible for this phenotype in EPEC. These findings suggest that efa1 encodes a novel virulence-associated determinant of AEEC, which contributes to the adhesive capacity of these bacteria.

The interstitial response to renal injury: fibroblast-like cells show phenotypic changes and have reduced potential for erythropoietin gene expression.

To define the potential for erythropoietin gene expression in injured kidneys, marker gene expression was examined in transgenic mice bearing a homologously recombined erythropoietin--simian virus 40 T antigen (Epo-TAg) transgene. Three types of renal injury were studied: ureteric obstruction, global ischemia following clamping of the renal pedicle, and focal needlestick injury. All modes of injury were associated with an expansion of the interstitial space, which contained an increased number of cells. Alterations observed in the interstitial fibroblast-like cells included an increased number and complexity of cellular processes, enhanced expression of contractile elements, particularly of the intermediate filament desmin, and reduced expression of ecto-5'-nucleotidase. Following each type of injury there was a focal or general reduction in the proportion of such cells that could be stimulated to express Epo-TAg. However, some positively staining cells were present even in severely injured regions and more could be recruited to express Epo-TAg by severe anemic or hypoxic stimulation, indicating that cells with the potential for erythropoietin gene expression were neither absent nor completely refractory to stimulation in these regions. In all injured kidneys, Epo-TAg expression was limited to the fibroblast-like population. Double labeling experiments showed that cells expressing Epo-TAg also expressed increased amounts of desmin, demonstrating that the myofibroblast features which develop in response to injury and the capacity for erythropoietin gene expression are not mutually exclusive.

Simplification and spin-spin analysis of the side chain proton magnetic resonance spectrum of the decapeptide gramicidin S using difference scalar decoupling and biosynthesis of specifically deuterated analogs.

Biosynthesis of specifically deuterated molecules and difference scalar decoupling permitted an analysis of all C alpha-C beta spin systems of gramicidin S. Proof is presented that proton magnetic resonance spectra obtained by difference scalar decoupling yield not only spectral assignments and simplification but also accurate chemicals shifts and scalar coupling constants. The variations in (3J alpha beta) and in proton chemical shifts at temperatures over the range of -54 degrees -+66 degrees C are consistent with the internal rotation around the C alpha-C beta bonds of Val1, Orn2, Leu3, and Phe4 residues discovered using carbon 13 spectroscopy. The value (3J alpha beta) = 1.5 Hz for the proline residue is consistent with there being only one C alpha-C beta conformer. This is supported by the small temperature dependence of (3J alpha beta). However, it cannot be rigorously excluded that oscillation between a major and a minor C alpha-C beta conformation occurs for proline.

Selection of mutant CHO cells with constitutive activation of the HIF system and inactivation of the von Hippel-Lindau tumor suppressor.

Hypoxia-inducible factor (HIF) mediates a widespread transcriptional response to hypoxia through binding to cis-acting DNA sequences termed hypoxia response elements (HREs). Activity of the transcriptional complex is suppressed in the presence of oxygen by processes that include the targeting of HIF-alpha subunits for ubiquitin-mediated proteolysis. To provide further insights into these processes we constructed Chinese hamster ovary (CHO) cells bearing stably integrated plasmids that expressed HRE-linked surface antigens and used these cells in genetic screens for mutants that demonstrated constitutive up-regulation of HRE activity. From mutagenized cultures, clones were isolated that demonstrated up-regulation of HRE activity and increased HIF-1alpha protein levels in normoxic culture. Transfection and cell fusion studies suggested that these cells possess recessive defects that affect one or more pathways involved in HIF-alpha proteolysis. Two lines were demonstrated to harbor truncating mutations in the von Hippel-Lindau (VHL) tumor suppressor gene. In these cells, defects in ubiquitylation of exogenous human HIF-1alpha in vitro could be complemented by wild type pVHL, and re-expression of a wild type VHL gene restored a normal pattern of HIF/HRE activity, demonstrating the critical dependence of HIF regulation on pVHL in CHO cells. In contrast, other mutant cells had no demonstrable mutation in the VHL gene, and ubiquitylated exogenous HIF-1alpha normally, suggesting that they contain defects at other points in the oxygen-regulated processing of HIF-alpha subunits.

Identification of the renal erythropoietin-producing cells using transgenic mice.

Regulation of erythropoietin production by the kidneys is central to the control of erythropoiesis. Uncertainty about the identity of the renal cells involved has been a major obstacle to understanding this mechanism. We have used sequence from the mouse erythropoietin locus to direct expression of a marker gene, SV40 T antigen, to these cells in transgenic mice. The transgenic constructs contained an oligonucleotide marker (Epo-M) or SV40 sequence (Epo-TAg) in the 5' untranslated region of the mouse erythropoietin gene, flanked on each side by 9 and 7.5 kb of DNA from the mouse erythropoietin locus. Anemia-inducible expression of Epo-M and Epo-TAg was observed in the kidney. In one of thirteen lines, homologous integration of Epo-TAg into the mouse erythropoietin locus occurred. In transgenic mice bearing Epo-TAg at homologous and heterologous insertion sites, renal expression was restricted to a population of cells in the interstitium of the cortex and outer medulla. Immunohistochemical characterization by light and electron microscopy shows that these are the fibroblast-like type I interstitial cells.

Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth.

Recent studies of tissue culture cells have defined a widespread system of oxygen-regulated gene expression based on the activation of a heterodimeric transcription factor termed hypoxia-inducible factor-1 (HIF-1). To determine whether the HIF-1 transcriptional response is activated within solid tumors and to define the consequences, we have studied tumor xenografts of a set of hepatoma (Hepa-1) cells that are wild type (wt), deficient (c4), and revertant (Rc4) for an obligatory component of the HIF-1 heterodimer, HIF-1beta. Because HIF-1beta is also essential for the xenobiotic response (in which it is termed the aryl hydrocarbon receptor nuclear translocator), we also studied c31 cells, which have a different defect in the xenobiotic response and form the HIF-1 complex normally. Two genes that show different degrees of HIF-1-dependent hypoxia-inducible expression in cell culture were selected for analysis-the glucose transporter, GLUT3, and vascular endothelial growth factor (VEGF). In situ hybridization showed intense focal induction of gene expression in tumors derived from wt, Rc4, and c31 cells, which was reduced (VEGF) or not seen (GLUT3) in those derived from c4 cells. In association with these changes, tumors of c4 cells had reduced vascularity and grew more slowly. These findings show that HIF-1 activation occurs in hypoxic regions of tumors and demonstrate a major influence on gene expression, tumor angiogenesis, and growth.