Using routinely collected data to stratify prostate cancer patients into phases of care in the United Kingdom: implications for resource allocation and the cancer survivorship programme.

BACKGROUND: Prostate cancer is the most commonly diagnosed malignancy in British men. The increasing use of PSA screening test has resulted in many more patients being diagnosed with this condition. Advances in its treatment have improved the survival rate among these patients. By 2040, the prevalence of prostate cancer survivors is expected to reach 830 000. Many of them will require medical support for the management of their progressive disease or long-term toxicities from previous treatments. Successful implementation of the cancer survivorship programme among these patients depends on a good understanding of their demand on the health care system. The aim of this study is to segment the population of prostate cancer survivors into different needs groups and to quantify them with respect to their phase of care. METHODS: Incidence, survival, prevalence and mortality data collected and reported by cancer registries across the United Kingdom have been used for the current study to provide indicative estimates as to the number of prostate cancer patients in each phase of the care pathway in a year. RESULTS: The majority of prostate cancer patients are in the post-treatment monitoring phase. Around a fifth of the patients are either receiving treatment or in the recovery and readjustment phase having completed their treatment in the preceding year. Thirteen percent have not received any anticancer treatment, a further 12% (32 000) have developed metastatic disease and 4% are in the final stage of their lives. CONCLUSION: On the basis of our estimates, patients undergoing post-treatment monitoring phase will constitute the biggest group among prostate cancer survivors. The pressure to provide adequate follow-up care to these patients will be a challenge. There is limited data available to definitively quantify the number of prostate cancer patients who follow different pathways of care, and we hope this study has highlighted the importance of collecting and reporting of such data to help future health care planning for these patients.

New pathways of care for cancer survivors: adding the numbers.

BACKGROUND: Two million people in the UK had a cancer diagnosis at the end of 2008. Understanding the number of people diagnosed with cancer with and without health needs is valuable information that can be used to inform service planning, treatment provision and support for people at the right time in the right place as demand grows over time. METHODS: Using available data and clinically led assumptions about patient need and outcomes, we make indicative estimates. We quantify, for three common cancers, the number of people in each of the five main identified phases of the cancer care pathway. RESULTS: Estimates are provided for each phase of the pathway for breast, colorectal and lung cancers. We estimate that there are nearly 575,000 women a year with breast cancer in the care pathway at some point in the year, 8% are in the rehabilitation phase and 4% in the progressive illness phase. This compares to nearly 270,000 with colorectal and around 95,000 with lung cancer. CONCLUSION: Using readily available data, we estimate the numbers of patients with different health needs. These numbers could inform the targeting of resources for service providers.

Early biochemical signals arise from low affinity TCR-ligand reactions at the cell-cell interface.

The kinetics of acid release by a mixture of T cells and antigen presenting cells were measured with a microphysiometer during a brief exposure to antigenic peptides. We find that some of the early biochemical events that lead to cellular proliferation cause a specific increase in the rate of acid release. The duration of this increase in acid release reflects the life-time of the peptide-MHC complexes. Peptides that form long-lived complexes produce a response that is stable for more than an hour. Serial TCR engagement is suggested by the observation that the amplitude of this stable response can be rapidly shifted up or down with additional agonist peptide or with antibodies that block T cell receptor binding. Cells briefly exposed to a peptide that forms short-lived peptide-MHC complexes produce a response that decays rapidly as peptide is washed away. A quantitative analysis of the kinetics of this decay in acidification demonstrates that intercellular TCR-ligand reactions are rapid, reversible, and of low apparent affinity with < 20% of peptide-MHC ligand bound to a TCR at any one time. These results demonstrate that the fraction of peptide-MHC ligands bound to TCRs at the cell-cell interface is no higher than anticipated from the affinities observed in solution for isolated TCRs and ligands.

Evidence that the autoimmune antigen myelin basic protein (MBP) Ac1-9 binds towards one end of the major histocompatibility complex (MHC) cleft.

The NH2-terminal peptide of myelin basic protein (MBP) bound to the class II major histocompatibility complex (MHC) protein I-Au is an immunodominant epitope in experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis. However, the MBP-I-Au complex is very unstable. To investigate this, we performed site-directed mutagenesis of the I-Au MHC protein and the MBP peptide. Biochemical, T cell activation, and molecular modeling studies of mutant complexes demonstrate that the MBP peptide's key residue for MHC binding, lysine 4, is buried in the P6 pocket of I-Au, which is predominantly hydrophobic. This implies that the MBP-I-Au complex differs from more stable complexes in two respects: (a) the peptide leaves the NH2-terminal region of the MHC peptide-binding cleft unoccupied; (b) the peptide is not anchored by typical favorable interactions between peptide side chains and MHC pockets. To test these hypotheses, a modified MBP peptide was designed based on molecular modeling, with the aim of producing strong I-Au binding. Extension of the NH2 terminus of MBP with six amino acids from the ova peptide, and replacement of the lysine side chain in the P6 pocket with an aromatic anchor, results in >1,000-fold increased binding stability. These results provide an explanation for the unusual peptide-MHC-binding kinetics of MBP, and should facilitate an understanding of why mice are not tolerant to this self-peptide- MHC complex.

Kinetics and extent of T cell activation as measured with the calcium signal.

We have characterized the calcium response of a peptide-major histocompatibility complex (MHC)-specific CD4(+) T lymphocyte line at the single cell level using a variety of ligands, alone and in combination. We are able to distinguish four general patterns of intracellular calcium elevation, with only the most robust correlating with T cell proliferation. Whereas all three antagonist peptides tested reduce the calcium response to an agonist ligand, two give very different calcium release patterns and the third gives none at all, arguing that (a) antagonism does not require calcium release and (b) it involves interactions that are more T cell receptor proximal. We have also measured the time between the first T cell-antigen-presenting cell contact and the onset of the calcium signal. The duration of this delay correlates with the strength of the stimulus, with stronger stimuli giving a more rapid response. The dose dependence of this delay suggests that the rate-limiting step in triggering the calcium response is not the clustering of peptide-MHC complexes on the cell surface but more likely involves the accumulation of some intracellular molecule or complex with a half-life of a few minutes.

Distinguishing Extravascular from Intravascular Ferumoxytol Pools within the Brain: Proof of Concept in Patients with Treated Glioblastoma.

BACKGROUND AND PURPOSE: Glioblastoma-associated macrophages are a major constituent of the immune response to therapy and are known to engulf the iron-based MR imaging contrast agent, ferumoxytol. Current ferumoxytol MR imaging techniques for localizing macrophages are confounded by contaminating intravascular signal. The aim of this study was to assess the utility of a newly developed MR imaging technique, segregation and extravascular localization of ferumoxytol imaging, for differentiating extravascular-from-intravascular ferumoxytol contrast signal at a delayed 24-hour imaging time point. MATERIALS AND METHODS: Twenty-three patients with suspected post-chemoradiotherapy glioblastoma progression underwent ferumoxytol-enhanced SWI. Segregation and extravascular localization of ferumoxytol imaging maps were generated as the voxelwise difference of the delayed (24 hours) from the early (immediately after administration) time point SWI maps. Continuous segregation and extravascular localization of ferumoxytol imaging map values were separated into positive and negative components. Image-guided biologic correlation was performed. RESULTS: Negative segregation and extravascular localization of ferumoxytol imaging values correlated with early and delayed time point SWI values, demonstrating that intravascular signal detected in the early time point persists into the delayed time point. Positive segregation and extravascular localization of ferumoxytol imaging values correlated only with delayed time point SWI values, suggesting successful detection of the newly developed extravascular signal. CONCLUSIONS: Segregation and extravascular localization of ferumoxytol MR imaging improves on current techniques by eliminating intrinsic tissue and intravascular ferumoxytol signal and may inform glioblastoma outcomes by serving as a more specific metric of macrophage content compared with uncorrected T1 and SWI techniques.

Identification of phagocytosis-associated surface proteins of macrophages by two-dimensional gel electrophoresis.

Two-dimensional PAGE (P. Z. O'Farrell, H. M. Goodman, and P. H. O'Farrell. 1977. Cell. 12:1133-1142) has been employed to assess the effects of antibody-dependent phagocytosis on the cell surface protein composition of RAW264 macrophages. Unilamellar phospholipid vesicles containing 1% dinitrophenyl-aminocaproyl-phosphatidylethanolamine (DNP-cap-PE) were used as the target particle. Macrophages were exposed to anti-DNP antibody alone, vesicles alone, or vesicles in the presence of antibody for 1 h at 37 degrees C. Cell surface proteins were then labeled by lactoperoxidase-catalyzed radioiodination at 4 degrees C. After detergent solubilization, membrane proteins were analyzed by two-dimensional gel electrophoresis. The resulting pattern of spots was compared to that of standard proteins. We have identified several surface proteins, not apparently associated with the phagocytic process, which are present either in a multichain structure or in several discretely charged forms. After phagocytosis, we have observed the appearance of two proteins of 45 and 50 kdaltons in nonreducing gels. In addition, we have noted the disappearance of a 140-kdalton protein in gels run under reducing conditions. These alterations would not be detected in the conventional one-dimensional gel electrophoresis. This evidence shows that phagocytosis leads to a modification of cell surface protein composition. Our results support the concept of specific enrichment and depletion of membrane components during antibody-dependent phagocytosis.

Disappearance of macrophage surface folds after antibody-dependent phagocytosis.

We have employed the method of Burwen and Satir (J. Cell Biol., 1977, 74:690) to measure the disappearance of surface folds from resident guinea pig peritoneal macrophages after antibody-dependent phagocytosis. Unilamellar phospholipid vesicles containing dimyristoylphosphatidylcholine and 1 mol % dinitrophenyl-epsilon-aminocaproyl-phosphatidylethanolamine, a lipid that possesses a hapten headgroup, were prepared by an ether injection technique. These vesicles were taken up by macrophages in a time- and temperature-dependent fashion. Vesicles that contained ferritin trapped in the internal aqueous volume were identified within macrophages by transmission electron microscopy. Scanning electron microscopy has shown that macrophage surface folds decrease dramatically after phagocytosis. The surface fold length (micrometer) per unit smooth sphere surface area (micrometer2) decreases from 1.3 +/- 0.3 micrometer-1 to 0.53 +/- 0.25 micrometer-1 when cells are incubated in the presence of specific anti-DNP antibody and vesicles at 37 degrees C. No significant effect was observed in the presence of antibody only or vesicles only. Our studies shown that phagocytosis is associated with a loss of cell surface folds and a loss of cell surface area, which is consonant with current views of the endocytic process. On the basis of our uptake data, we estimate that approximately 400 micrometer2 of vesicle surface membrane is internalized. The guinea pig macrophage plasma membrane has a total area of approximately 400 micrometer2 in control studies, whereas the cells have roughly 300 micrometer2 after phagocytosis. These estimates of surface areas include membrane ruffles and changes directly related to changes in cell volume. We suggest that during antibody-dependent phagocytosis a membrane reservoir is made available to the cell surface.

Lipid monolayer-coated solid surfaces do not perturb the lateral motion and distribution of C3b receptors on neutrophils.

We have used epifluorescence and photobleaching techniques to study the lateral distribution and motion of fluorescein-conjugated Fab fragments of anti-C3b receptor antibody bound to human neutrophils when the cells rest on various solid supports (microscope slides or cover slips). Supports composed of quartz, glass, or alkylated glass induced cellular adhesion, spreading, and an extensive lateral redistribution of C3b receptors (but not HLA antigens). The neutrophil C3b receptors become patchy, and the patches apparently undergo nonrandom translational motion. Many patches are found on the upper surfaces of the cells removed from the region of cell membrane-glass contact. In contrast, neutrophils supported by lipid monolayer-coated glass do not adhere or spread, and the C3b receptor remains uniform and diffuses freely (D approximately equal to 2 X 10(-10) cm2/s).

Energy transfer between two peptides bound to one MHC class II molecule.

The 17-amino acid peptide from chicken ovalbumin, Ova(323-339), was labeled at the amino terminus with fluorescein [FOva(323-339)] and near the carboxyl terminus with Texas Red [AcOva(323-338)KTR]. Fluorescence spectroscopy was carried out on resolved electrophoretic bands on nonreducing polyacrylamide gels derived from incubation mixtures containing major histocompatibility complex (MHC) class II molecules IAd and the FOva(323-339)- and AcOva(323-338)KTR-labeled peptides. Energy transfer between fluorescein and Texas Red was observed in the "floppy" alpha beta heterodimer band, but not in the "compact" alpha beta heterodimer band. Energy transfer was detected between the truncated peptides FOva(323-328)CONH2 and AcOva(331-338)KTR in both the compact alpha beta and floppy alpha beta gel bands. The energy-transfer data suggest that the two binding sites of floppy alpha beta arise from splitting apart a putative large, single binding site region in compact alpha beta.

Electric field-induced concentration gradients in lipid monolayers.

Externally applied electric field gradients gave rise to lateral concentration gradients in monolayers of certain binary lipid mixtures. For binary mixtures of dihydrocholesterol and dimyristoylphosphatidylcholine, the application of an electric field gradient at pressures below the critical pressure produced a liquid-liquid phase separation in a monolayer that is otherwise homogenous. At pressures slightly above the critical pressure, a field gradient produced a large concentration gradient without phase separation. The lipid concentration gradients can be described by equilibrium thermodynamic chemical potentials. The observed effects appear to be relevant to the structure and composition of biological membranes.

Light-addressable potentiometric sensor for biochemical systems.

Numerous biochemical reactions can be measured potentiometrically through changes in pH, redox potential, or transmembrane potential. An alternating photocurrent through an electrolyte-insulator-semiconductor interface provides a highly sensitive means to measure such potential changes. A spatially selectable photoresponse permits the determination of a multiplicity of chemical events with a single semiconductor device.

Neutrophil activation monitored by flow cytometry: stimulation by phorbol diester is an all-or-none event.

The population dynamics of single-cell stimulation was analyzed by monitoring autofluorescence by flow cytometry. Stimulation of the respiratory burst in human neutrophils by 12-O-tetradecanoyl phorbol-13-acetate (TPA) caused a decline in highly fluorescent cells (characteristic of resting neutrophils) and a corresponding increase in the number of weakly fluorescent cells (characteristic of activated neutrophils). Increasing concentrations of TPA caused increasing numbers of cells to shift from the highly fluorescent population to the weakly fluorescent population without the appearance of intermediate populations. Thus the neutrophil respiratory burst, a component of neutrophil cytotoxic response, is triggered in an all-or none fashion.

The cytosensor microphysiometer: biological applications of silicon technology.

A silicon-based device, dubbed a microphysiometer, can be used to detect and monitor the response of cells to a variety of chemical substances, especially ligands for specific plasma membrane receptors. The microphysiometer measures the rate of proton excretion from 10(4) to 10(6) cells. This article gives an overview of experiments currently being carried out with this instrument with emphasis on receptors with seven transmembrane helices and tyrosine kinase receptors. As a scientific instrument, the microphysiometer can be thought of as serving two distinct functions. In terms of detecting specific molecules, selected biological cells in this instrument serve as detectors and amplifiers. The microphysiometer can also investigate cell function and biochemistry. A major application of this instrument may prove to be screening for new receptor ligands. In this respect, the microphysiometer appears to offer significant advantages over other techniques.

Detection of cell-affecting agents with a silicon biosensor.

Cellular metabolism is affected by many factors in a cell's environment. Given a sufficiently sensitive method for measuring cellular metabolic rates, it should be possible to detect a wide variety of chemical and physical stimuli. A biosensor has been constructed in which living cells are confined to a flow chamber in which a potentiometric sensor continually measures the rate of production of acidic metabolites. Exploratory studies demonstrate several applications of the device in basic science and technology.

Critical pressures in multicomponent lipid monolayers.

Epifluorescence microscopy has been used previously to study coexisting liquid phases in lipid monolayers of dihydrocholesterol and dimyristoylphosphatidylcholine at the air/water interface. This binary mixture has a critical point at room temperature (22 degrees C), a monolayer pressure of approx. 10 mN/m, and a composition in the vicinity of 20-30 mol% dihydrocholesterol. It is reported here that this critical pressure can be lowered, raised, or maintained constant by systematically replacing molecules of this phosphatidylcholine with molecules of a phosphatidylethanolamine, or an unsaturated phosphatidylcholine, or mixtures of the two, while maintaining the dihydrocholesterol concentration at 20 mol%. Thus, even complex mixtures of lipids may be characterized by a single, well-defined second-order phase transition. In principle, such transitions might be found in biological membranes.

Cytochemical study of liposome and lipid vesicle phagocytosis.

Electron microscopy cytochemistry has been used to study the cytoplasmic location of liposomes and lipid vesicles following specific antibody-dependent phagocytosis. The vesicle compositions were 94-99 mol% 'fluid' lipid (egg phosphatidylcholine or dimyristoylphosphatidylcholine at 37 degrees C or 'solid' lipid (dipalmitoylphosphatidylcholine at 37 degrees C). In some cases, 4 mol% phosphatidylserine was included in the vesicle membrane so as to vary the surface charge density. These vesicles undergo specific antibody-dependent phagocytosis by RAW264 macrophages when the lipid membranes contain 1-2 mol% dinitrophenyl lipid hapten in the presence of rabbit anti-dinitrophenyl IgG antibody. Internalized lipid vesicles can be visualized with the electron microscope when ferritin is trapped in the internal aqueous compartments prior to internalization. The lipid vesicles were demonstrated to be internal to the macrophage plasma membranes by selectively staining the plasma membranes with Ruthenium red. The cytoplasmic location of vesicles and liposomes was studied by electron microscopic staining for activities of the following enzymes: (1) acid phosphatase; (2) inorganic trimetaphosphatase; (3) adenosine triphosphatase; and (4) glucose-6-phosphatase. The first two enzymatic activities were found in association with ferritin-containing vesicles after antibody-dependent phagocytosis, showing the formation of vesicle-containing phagolysosomes. Adenosine triphosphatase and glucose-6-phosphatase were primary not associated with the vesicles, suggesting a minimal association of vesicles with plasma membrane, Golgi, endoplasmic reticulum and perinuclear cisternae. Phagosome-lysosome fusion did not appear to depend on the type of target lipid vesicle or liposome, on the 'fluidity' of the target membrane, or the presence of phosphatidylserine in the target membrane.

Multiple phase equilibria in binary mixtures of phospholipids.

Approximate phse diagrams describing lateral phase separations are given for binary mixtures of dimyristoyl phosphatidylcholine with dipalmitoyl phosphatidylcholine, distearoyl phosphatidycholine, and dipalmitoyl phosphatidylethanolamine. These diagrams are based in part on freeze-fracture electron microscopic data. These phase diagrams represent an improvement over previous studies in that both solid phses (Pbeta' and Lbeta') of the phosphatidylcholines are included. Further consideration is given to the problem of binary mixtures in which there are two Pbeta' phases that do not form a continuous range of solid solutions.

Lateral phase separations in binary mixtures of phospholipids having different charges and different crystalline structures.

Synthetic dipalmitoyl phosphatidylserine exhibits a sharp chain-melting transition temperature at 51 degrees C as judged by partitioning of the spin label 2,2,6,6-tetramethylpiperidine-1-oxyl. Phase diagrams representing lateral phase separations in binary mixtures of dipalmitoyl phosphatidylserine with dipalmitoyl phosphatidylcholine as well as with dimyristoyl phosphatidylcholine are derived from paramagnetic resonance determinations of 2,2,6,6,-tetramethylpiperidine-1-oxyl partitioning, freeze-fracture electron microscopic studies and theoretical arguments that limit the general form of acceptable phase diagrams. The reported phase diagrams are the first to describe binary mixtures in which one lipid is charged and the second lipid uncharged. These phase diagrams also are the first to include the problem of solid phases with different crystalline conformations as it relates to the occurrence of a pretransition in phosphatidylcholines and its absence in phosphatidylserines. In addition to the phase diagrams reported here for these two binary mixtures, a brief theoretical discussion is given of other possible phase diagrams that may be appropriate to other lipid mixtures with particular consideration given to the problem of crystalline phases of different structures and the possible occurrence of second-order phase transitions in these mixtures.

The intermediate monoclinic phase of phosphatidylcholines.

Two pure phospholipids, dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, have been studied using freeze-fracture electron microscopy and the partitioning of the spin label, TEMPO. It is found that the characteristic band pattern, corresponding to monoclinic symmetry in multilamellar liposomes, is observed only in freeze-fracture electron microphotographs when samples are quenched from temperatures intermediate between the chain melting transition temperature and the pretransition temperature of the membrane. Markings are also observed on fracture faces of samples quenched from below the pretransition, but these "bands" are few in number and are widely and irregularly spaced. The lipid membranes used for freeze-fracture were prepared using detergent dialysis and are thought to consist of one, two, or some small number of concentric bilayer shells. These observations are in excellent accord with the recent, prior studies of Janiak, M.J., Small, D.M. and Shirley, G.G., ((1976) Biochemistry 15, 4575--4580), who found monoclinic symmetry (Pbeta' structure) in multilamellar liposomes of these phospholipids only when the sample temperature was intermediate between the main, chain melting transition temperature, and the pretransition temperature. The significance of these results for relating freeze-fracture electron microphotographis to phase diagrams derived from spin label or calorimetric data is discussed briefly. 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) partitioning data show distinct differences between liposomal preparations of these lipids, and other preparations having fewer bilayers per vesicular structure, with respect to the position, width, and hysteresis of the pretransition.