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.

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.

Baseline hydrocarbon levels in New Zealand coastal and marine avifauna.

The external effects of oil on wildlife can be obvious and acute. Internal effects are more difficult to detect and can occur without any external signs. To quantify internal effects from oil ingestion by wildlife during an oil spill, baseline levels of ubiquitous hydrocarbon fractions, like polycyclic aromatic hydrocarbons (PAHs), need to be established. With these baseline values the extent of impact from exposure during a spill can be determined. This research represents the first investigation of baseline levels for 22 PAHs in New Zealand coastal and marine avian wildlife. Eighty-five liver samples were tested from 18 species. PAHs were identified in 98% of livers sampled with concentrations ranging from 0 to 1341.6 ng/g lipid wt or on wet wt basis, 0 to 29.5 ng/g. Overall, concentrations were low relative to other globally reported avian values. PAH concentration variability was linked with species foraging habitat and migratory patterns.

Pharmacokinetics of oxycodone after subcutaneous administration in a critically ill population compared with a healthy cohort.

This study aimed to characterise and compare the absorption pharmacokinetics of a single subcutaneous dose of oxycodone in critically ill patients and healthy subjects. Blood samples taken at intervals from two minutes to eight hours after a subcutaneous dose of oxycodone in patients (5 mg) and healthy volunteers (10 mg) were assayed using high performance liquid chromatography. Data were analysed using a non-compartmental approach and presented as mean (SD). Parameters were corrected for dose differences between the groups assuming linear kinetics. Ten patients (eight male, two female) and seven healthy male subjects were included. Maximum venous concentration and area under the concentration curve were approximately two-fold lower in the patient group for an equivalent dose, suggesting either reduced bioavailability or increased clearance: maximum venous concentration 0.14 ± 0.06 vs 0.05 ± 0.02 µg/ml (P <0.0001); area under the concentration curve 19.50 ± 9.15 vs 9.72 ± 2.71 µg/ml/minute (P <0.001) respectively. However, time to maximum venous concentration and mean residence time were not different, suggesting similar absorption rates: time to maximum venous concentration 22.10 ± 18.0 vs 20.50 ± 16.10 minutes (P=0.81); mean residence time 353 ± 191 vs 291 ± 80 minutes (P=0.26). Kinetic parameters were less variable in patients than in volunteers. The patients therefore had reduced exposure to subcutaneous oxycodone. This warrants further model-based analysis and experimentation. Dose regimens for subcutaneous oxycodone developed in healthy volunteers cannot be directly translated to critically ill 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.

Contrast inversion in the epifluorescence of cholesterol-phospholipid monolayers.

A number of dihydrocholesterol-phospholipid mixtures have been examined using the epifluorescence microscopy of monolayers at the air-water interface. These mixtures form two coexisting liquids. Fluorescence contrast was provided using a variety of different lipid probes. With increasing monolayer pressure, all of the charged probes show contrast inversion at higher dihydrocholesterol concentrations. That is, with increasing pressure the charged probes transfer from one liquid to the other, reversing the fluorescence contrast. A wide variety of phospholipids were studied, and the inversion was seen in all cases. In the inverted state and at the higher dihydrocholesterol concentrations, the immiscibility persists to the highest pressures employed, 30-40 mN/m. The data show that binary dihydrocholesterol-phospholipid mixtures can form three distinct liquids, one of which is interpreted as a phase rich in condensed complex.

Rheumatoid arthritis (RA)-associated HLA-DR alleles form less stable complexes with class II-associated invariant chain peptide than non-RA-associated HLA-DR alleles.

Certain HLA-DR alleles confer strong susceptibility to the autoimmune disease rheumatoid arthritis (RA). We compared RA-associated alleles, HLA-DR*0401, HLA-DR*0404, and HLA-DR*0405, with closely related, non-RA-associated alleles, HLA-DR*0402 and HLA-DR*0403, to determine whether they differ in their interactions with the class II chaperone, invariant chain (Ii). Ii binds to class II molecules in the endoplasmic reticulum, inhibits binding of other ligands, and directs class II-Ii complexes to endosomes, where Ii is degraded to class II-associated Ii peptide (CLIP). To evaluate the interaction of Ii and CLIP with these DR4 alleles, we introduced HLA-DR*0401, *0402, and *0404 alleles into a human B cell line that lacked endogenous HLA-DR or HLA-DM molecules. In a similar experiment, we introduced HLA-DR*0403 and *0405 into an HLA-DM-expressing B cell line, 8.1.6, and its DM-negative derivative, 9.5.3. Surface abundance of DR4-CLIP peptide complexes and their susceptibility to SDS-induced denaturation suggested that the different DR4-CLIP complexes had different stabilities. Pulse-chase experiments showed CLIP dissociated more rapidly from RA-associated DR molecules in B cell lines. In vitro assays using soluble rDR4 molecules showed that DR-CLIP complexes of DR*0401 and DR*0404 were less stable than complexes of DR*0402. Using CLIP peptide variants, we mapped the reduced CLIP interaction of RA-associated alleles to the shared epitope region. The reduced interaction of RA-associated HLA-DR4 molecules with CLIP may contribute to the pathophysiology of autoimmunity in RA.

Condensed complexes and the calorimetry of cholesterol-phospholipid bilayers.

A recent thermodynamic model describes a reversible reaction between cholesterol (C) and phospholipid (P) to form a condensed complex C(nq)P(np). Here q and p are relatively prime integers used to define the stoichiometric composition, and n is a measure of cooperativity. The present study applies this model to the scanning calorimetry of binary mixtures of cholesterol and saturated phosphatidylcholines, especially work by McElhaney and collaborators. These mixtures generally show two heat capacity peaks, a sharp peak and a broad peak. The sharp heat absorption is largely due to the chain melting transition of pure phospholipid. In the present work the broad heat absorption is attributed to the thermal dissociation of complexes. The best fits of the model to the data require the complex formation to be highly cooperative, with cooperativity n = 12. Detailed comparisons are made between model calculations and calorimetric data. A number of unusual features of the data arise naturally in the model. The principal discrepancy between the calculations and experimental results is a spurious calculated heat absorption peak. This discrepancy is related to the reported relative magnitudes of the integrated broad and sharp heat absorption curves.

Kinetics of registry selection of chimeric peptides binding to MHC II.

Major histocompatability complex type II proteins (MHC II) are alphabeta-heterodimeric glycoproteins that present peptides to the T cell receptor (TCR) of CD4(+) T-cells. This presentation may result in activation of these T-cells, depending on the nature of the peptide. Peptides interact specifically with MHC II with nine peptide amino acid positions, and the corresponding MHC II pocket positions are usually labeled P1-P9. However, the length of peptides binding to MHC II may be greater than nine amino acids, and therefore these peptides may potentially bind to the MHC II in more than one registry. To investigate the mechanism by which a long peptide binds to I-E(k), a murine MHC II, a chimeric peptide with two nonoverlapping registries, f-IAYLKQATKQLRMATPLLMR was designed. The IAYLKQATK peptide segment is based on moth cytochrome c 95-103 (MCC 95-103), and the QLRMATPLLMR segment is based on murine Ii CLIP 89-99 M90L (Ii CLIP 89-99 M90L). This chimeric peptide forms two isomeric complexes. The MCC and Ii CLIP registries dissociate from I-E(k) with t(1/2) values of >>800 and 4.94 h, respectively. The registry composition of this MHC II/chimeric peptide complex was found to change as a function of time in approaching thermodynamic equilibrium: the results are consistent with a kinetic model that involves no intramolecular isomer interconversion. The model depicts uncorrelated binding to the MHC II determined by relative association rates to the two registries. This is followed by dissociation and subsequent rebinding, leading ultimately to a preponderance of the most stable complex. Similar results were obtained at pH 5.3. The behavior of this chimeric peptide approximates the binding of a 1:1 solution mixture of two peptides to MHC II, where the more stable complex is selected over time. We have also found that a chimeric peptide and a human MHC II, HLA-DR40401, form isomers with relative association rates to DR0401 at pH 5.3 of 15% for one isomer and 85% for the second isomer.

Peptide binding to active class II MHC protein on the cell surface.

Solution studies have demonstrated the existence of two functionally distinct isomers of empty class II MHC: an active isomer that binds peptide and an inactive isomer that does not. Empty MHC molecules on the surface of APCs can load antigenic peptides directly from the extracellular medium, facilitating the generation of a diverse peptide repertoire for T cell presentation. In this report, we examine I-Ek on the surface of Chinese hamster ovary cells with respect to the active and inactive isomers. As in the case of purified soluble active I-Ek, active I-Ek on the cell surface is unstable, decaying to the inactive form in approximately 14 min. Evidence is presented suggesting that at steady state <1% of the total cell surface I-Ek is active and that a significant fraction of these active molecules originates from intracellular pools as well as reactivation of inactive cell surface I-EK.

Stoichiometry of cholesterol-sphingomyelin condensed complexes in monolayers.

Some binary mixtures of cholesterol and phospholipids in monolayers have thermodynamic phase diagrams with two upper miscibility critical points. This feature has been interpreted in terms of 'condensed complexes' between the phospholipid and cholesterol. The present work gives evidence for the formation of complexes with a common simple integral stoichiometry in binary mixtures of cholesterol and a series of five sphingomyelins where the amide-linked acyl chain length is varied. This indicates that these complexes have a distinct geometry even though they form a liquid phase.

pH stability of HLA-DR4 complexes with antigenic peptides.

Complexes between antigenic peptides and class II proteins of the major histocompatibility complex (MHC) trigger cellular immune responses. These complexes usually dissociate more rapidly at mildly acidic pH, where they are formed intracellularly, as compared to neutral pH, where they function at the cell surface. This paper describes the pH dependence of the dissociation kinetics of complexes between MHC proteins and antigenic peptides containing aspartic and glutamic acid residues. Some of these complexes show an unusual pH dependence, dissociating much more rapidly at pH 7 than at pH 5.3. This occurs when the carboxylate group of the aspartic or glutamic acid residue is located in a neutral pocket of the protein. In contrast, solvent-exposed carboxylate groups or carboxylate groups buried in pockets where they form salt bridges with the protein do not show this unusual pH dependence. The kinetic data having the unusual pH dependence conform closely to a model in which there is a rapid reversible equilibration between a less stable deprotonated complex and a more stable protonated complex. In this model, the pK(a) of the protonation reaction for the partially buried peptide carboxylate group ranges from 7.7 to 8.3, reflecting the strongly basic conditions required for deprotonation. One of the few peptide/MHC complexes demonstrated to play a role in autoimmunity in humans contains a buried peptide carboxylate and shows this unusual pH dependence. The relevance of this finding to understanding the chemical basis of autoimmunity is briefly discussed.

Psychotropic interactions with warfarin.

OBJECTIVE: Improving knowledge about the cytochrome p450 system means that potential drug interactions can be predicted. Interactions involving warfarin may be thus avoidable. As many patients who have suffered from a stroke or other thromboembolic events may also develop psychiatric disorder, knowledge about possible interactions with psychotropics is essential for prescribers. METHOD: A Medline and hand search of published literature was complemented by contacting manufacturers. RESULTS: The antidepressants citalopram, nefazodone and sertraline have relatively low interaction potential with warfarin; fluoxetine and fluvoxamine relatively high. Carbamazepine appears to reduce warfarin's anticoagulant effect. Other antipsychotics, antidepressants and anxiolytics have only a theoretical risk of interaction. Lithium, gabapentin, sulpiride and amisulpride are predominantly renally excreted and so are not likely to interact with warfarin. CONCLUSION: Many psychotropics are involved in predictable interactions with warfarin. Drugs with a known low interaction potential should be chosen instead of those known or predicted to interact.

Miscibility critical pressures in monolayers of ternary lipid mixtures.

When phospholipids are mixed with cholesterol in a monolayer at an air-water interface, coexisting 2-dimensional liquid phases can be observed if the surface pressure, pi, is lower than the miscibility critical pressure, pi(c). Ternary mixtures of two phospholipid species with dihydrocholesterol have been reported to have critical pressures that are linearly proportional to the relative composition of the phospholipids. However, we report here that, if the acyl chains of the two phospholipids differ significantly in length or unsaturation, the behavior is markedly different. In this case, the critical pressure of the ternary mixture can be remarkably high, exceeding the critical pressures of the corresponding binary mixtures. High critical pressures are also seen in binary mixtures of phospholipid and dihydrocholesterol when the two acyl chains of the phospholipid differ sufficiently in length. Using regular solution theory, we interpret the elevated critical pressures of these mixtures as an attractive interaction between the phospholipid components.

Condensed complexes, rafts, and the chemical activity of cholesterol in membranes.

Epifluorescence microscopy studies of mixtures of phospholipids and cholesterol at the air-water interface often exhibit coexisting liquid phases. The properties of these liquids point to the formation of "condensed complexes" between cholesterol and certain phospholipids, such as sphingomyelin. It is found that monolayers that form complexes can incorporate a low concentration of a ganglioside G(M1). This glycolipid is visualized by using a fluorescently labeled B subunit of cholera toxin. Three coexisting liquid phases are found by using this probe together with a fluorescent phospholipid probe. The three liquid phases are identified as a phospholipid-rich phase, a cholesterol-rich phase, and a condensed complex-rich phase. The cholera toxin B labeled ganglioside G(M1) is found exclusively in the condensed complex-rich phase. Condensed complexes are likely present in animal cell membranes, where they should facilitate the formation of specialized domains such as rafts. Condensed complexes also have a major effect in determining the chemical activity of cholesterol. It is suggested that this chemical activity plays an essential role in the regulation of cholesterol biosynthesis. Gradients in the chemical activity of cholesterol should likewise govern the rates and direction of intracellular intermembrane cholesterol transport.

Chemical activity of cholesterol in membranes.

Measurements are reported for the rate constants for the release of cholesterol (and dihydrocholesterol) to beta-cyclodextrin from mixtures with phospholipids in homogeneous monolayers at constant pressure at the air-water interface. In each mixture, it is found that the release rate shows a sharp decrease as the cholesterol concentration in the monolayer decreases through a composition corresponding to the stoichiometry of a cholesterol-phospholipid complex. The stoichiometry of the complex was established previously by the position of a sharp cusp in the thermodynamic phase diagram of each mixture and also by a minimum in average molecular area versus composition measurements. A theoretical model used earlier to account for the phase diagrams predicts the chemical potential and chemical activity of cholesterol in these mixtures. The calculated chemical activity also shows a sharp change at the complex stoichiometry in homogeneous monolayers. The similarities in change of observed release rate and calculated chemical activity are expected from reaction rate theory where the release rate is proportional to the cholesterol chemical activity. The chemical activity of cholesterol as determined by complex formation between some phospholipids and cholesterol in the plasma membrane of cells may serve a regulatory function with respect to intracellular cholesterol transport and biosynthesis.

Kinetics of peptide binding to the class II MHC protein I-Ek.

Class II MHC glycoproteins bind short (7-25 amino acid) peptides in an extended type II polyproline-like conformation and present them for immune recognition. Because empty MHC is unstable, measurement of the rate of the second-order reaction between peptide and MHC is challenging. In this report, we use dissociation of a pre-bound peptide to generate the active, peptide-receptive form of the empty class II MHC molecule I-Ek. This allows us to measure directly the rate of reaction between active, empty I-Ek and a set of peptides that vary in structure. We find that all peptides studied, despite having highly variable dissociation rates, bind with similar association rate constants. Thus, the rate-limiting step in peptide binding is minimally sensitive to peptide side-chain structure. An interesting complication to this simple model is that a single peptide can sometimes bind to I-Ek in two kinetically distinguishable conformations, with the stable peptide-MHC complex isomer forming much more slowly than the less-stable one. This demonstrates that an additional free-energy barrier limits the formation of certain specific MHC-peptide complex conformations.

Electric field effect on cholesterol-phospholipid complexes.

Monolayer mixtures of dihydrocholesterol and phospholipids at the air-water interface are used to model membranes containing cholesterol and phospholipids. Specific, stoichiometric interactions between cholesterol and some but not all phospholipids have been proposed to lead to the formation of condensed complexes. It is reported here that an externally applied electric field of the appropriate sign can destabilize these complexes, resulting in their dissociation. This is demonstrated through the application of an electric field gradient that leads to phase separations in otherwise homogeneous monolayers. This is observed only when the monolayer composition is close to the stoichiometry of the complex. The electric field effect is analyzed with the same mean field thermodynamic model as that used previously to account for pairs of upper miscibility critical points in these mixtures. The concentrations of dihydrocholesterol, phospholipid, and complex vary strongly and sometimes discontinuously in the monolayer membrane in the field gradient. The model is an approximation to a two-dimensional liquid in which molecules freely exchange between free and complexed form so that the chemical potentials are constant throughout the membrane. The calculations are illustrated for a complex of about 15 molecules, composed of 5 cholesterol molecules and 10 phospholipid molecules.

Interpretation of biphasic dissociation kinetics for isomeric class II major histocompatibility complex-peptide complexes.

Antigenic peptides bound to class II major histocompatibility complex (MHC) proteins play a key role in the distinction between "self" and "nonself" by the cellular immune system. Although the formation and dissociation of these complexes are often thought of in terms of the simple mechanism [formula in text], studies of MHC-peptide dissociation kinetics suggest that multiple interconverting forms of the bound MHC-peptide complex can be formed. However, the precise relationship between observed dissociation data and proposed multiple-complex mechanisms has not been systematically examined. Here we provide a mathematical analysis to fill this gap and attempt to clarify the kinetic behavior that is expected to result from the proposed mechanisms. We also examine multiple-complex dynamics that can be "hidden" in conventional experiments. Although we focus on MHC-peptide interactions, the analysis provided here is fully general and applies to any ligand-receptor system having two distinct bound states.

Condensed complexes of cholesterol and phospholipids.

Mixtures of dihydrocholesterol and phospholipids form immiscible liquids in monolayer membranes at the air-water interface under specified conditions of temperature and 2-dimensional pressure. In recent work it has been discovered that a number of these mixtures exhibit two upper miscibility critical points. Pairs of upper critical points can be accounted for by a theoretical model that implies the cooperative formation of molecular complexes of dihydrocholesterol and phospholipid molecules. These complexes are calculated to be present in the membranes both above and below the critical points. Below the critical points the complexes form a separate phase, whereas above the critical points the complexes are completely miscible with the other lipid components. The cooperativity of complex formation prompts the use of the terminology condensed complex.