Angiotensin II Treatment Induces Reorganization and Changes in the Lateral Dynamics of Angiotensin II Type 1 Receptor in the Plasma Membrane Elucidated by Photoactivated Localization Microscopy Combined with Image Spatial Correlation Analysis.

The mechanisms by which angiotensin II type 1 receptor is distributed and the diffusional pattern in the plasma membrane (PM) remain unclear, despite their crucial role in cardiovascular homeostasis. In this work, we obtained quantitative information of angiotensin II type 1 receptor (AT1R) lateral dynamics as well as changes in the diffusion properties after stimulation with ligands in living cells using photoactivated localization microscopy (PALM) combined with image spatial-temporal correlation analysis. To study the organization of the receptor at the nanoscale, expansion microscopy (ExM) combined with PALM was performed. This study revealed that AT1R lateral diffusion increased after binding to angiotensin II (Ang II) and the receptor diffusion was transiently confined in the PM. In addition, ExM revealed that AT1R formed nanoclusters at the PM and the cluster size significantly decreased after Ang II treatment. Taking these results together suggest that Ang II binding and activation cause reorganization and changes in the dynamics of AT1R at the PM.

A correlative super-resolution protocol to visualise structural underpinnings of fast second-messenger signalling in primary cell types.

Nanometre-scale cellular information obtained through super-resolution microscopies are often unaccompanied by functional information, particularly transient and diffusible signals through which life is orchestrated in the nano-micrometre spatial scale. We describe a correlative imaging protocol which allows the ubiquitous intracellular second messenger, calcium (Ca2+), to be directly visualised against nanoscale patterns of the ryanodine receptor (RyR) Ca2+ channels which give rise to these Ca2+ signals in wildtype primary cells. This was achieved by combining total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals, with the subsequent DNA-PAINT imaging of the RyRs. We report a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets and demonstrate how such data can be used to visually identify the ensembles of Ca2+ channels that are locally activated during the genesis of cytoplasmic Ca2+ signals.

Investigating the Test of Premorbid Functioning (TOPF) in predicting Wechsler Abbreviated Scale of Intelligence - Second edition (WASI-II) scores in an Australian sample.

Accurate prediction of premorbid functioning is important in neuropsychological assessment. We aimed to investigate the predictive accuracy of the TOPF and examine this word list at an item level against WASI-II scores, using Australian pronunciations. The sample of 219 healthy Australians were aged 18-82 years. Multiple regression analyses were used to replicate the TOPF and simple demographic models based on the US TOPF standardization. Rasch analyses provided a comparison of Australian, US and UK word order from the proportion of words pronounced correctly. The variance explained in WASI-II index scores ranged from R2=.12 (PRI) to .33 (FSIQ-2), which was approximately half that reported in the US standardization study. The accuracy of predicted WASI-II scores was also slightly less in our sample. Thirty-two words were out of place by five places or more compared with the US word order and 30 compared with the UK. These results add to concerns about the application the TOPF with norms developed in the US and UK in the Australian context. Clinicians are advised not to apply the five error discontinue rule when using the TOPF in the local context. Development of a more accurate word reading task for use in Australia is warranted.

The factor structure of the Quality of Life Inventory (QOLI) following traumatic brain injury.

Quality of life is a key indicator of outcome following traumatic brain injury (TBI). Research has reported several different factor structures for the Quality of Life Inventory (QOLI, Frisch, 1994). We compared the fit of existing factor models and examined the clinical utility of the QOLI's factors in a sample of Australian adults with TBI. Archival data from 901 participants were provided by the Neurotrauma Register of Tasmania. Participants were aged 16-80 years and 63% were male. Approximately 69% had mild TBI (PTA < 24 h), approximately 24% had moderate TBI (PTA >1 day, <7 days) and 7% had severe TBI. Both cross sectional and longitudinal analyses were utilized, as participants provided data at one or more of seven time-points, up to 3 years following injury. The results showed the data best fitted a three-factor model, comprising Self-functioning and activity, Self-actualization and Family and environment factors, and a second order Overall QOL factor. Differences in the trajectory of recovery were noted between the QOLI factor scores over time and in relation to demographic and injury variables. In conclusion, the three-factor structure of the QOLI provided useful clinical information about the recovery of patients' subjective quality of life following TBI.

3D ultrastructural organisation of calcium release units in the avian sarcoplasmic reticulum.

Excitation-contraction coupling in vertebrate hearts is underpinned by calcium (Ca2+) release from Ca2+ release units (CRUs). CRUs are formed by clusters of channels called ryanodine receptors on the sarcoplasmic reticulum (SR) within the cardiomyocyte. Distances between CRUs influence the diffusion of Ca2+, thus influencing the rate and strength of excitation-contraction coupling. Avian myocytes lack T-tubules, so Ca2+ from surface CRUs (peripheral couplings, PCs) must diffuse to internal CRU sites of the corbular SR (cSR) during centripetal propagation. Despite this, avian hearts achieve higher contractile rates and develop greater contractile strength than many mammalian hearts, which have T-tubules to provide simultaneous activation of the Ca2+ signal through the myocyte. We used 3D electron tomography to test the hypothesis that the intracellular distribution of CRUs in the avian heart permits faster and stronger contractions despite the absence of T-tubules. Nearest edge-edge distances between PCs and cSR, and geometric information including surface area and volume of individual cSR, were obtained for each cardiac chamber of the white leghorn chicken. Computational modelling was then used to establish a relationship between CRU distance and cell activation time in the avian heart. Our data suggest that cSR clustered close together along the Z-line is vital for rapid propagation of the Ca2+ signal from the cell periphery to the cell centre, which would aid in the strong and fast contractions of the avian heart.

Pump-Less, Recirculating Organ-on-Chip (rOoC) Platform to Model the Metabolic Crosstalk between Islets and Liver.

Type 2 diabetes mellitus (T2DM), obesity, and metabolic dysfunction-associated steatotic liver disease (MASLD) are epidemiologically correlated disorders with a worldwide growing prevalence. While the mechanisms leading to the onset and development of these conditions are not fully understood, predictive tissue representations for studying the coordinated interactions between central organs that regulate energy metabolism, particularly the liver and pancreatic islets, are needed. Here, a dual pump-less recirculating organ-on-chip platform that combines human pluripotent stem cell (sc)-derived sc-liver and sc-islet organoids is presented. The platform reproduces key aspects of the metabolic cross-talk between both organs, including glucose levels and selected hormones, and supports the viability and functionality of both sc-islet and sc-liver organoids while preserving a reduced release of pro-inflammatory cytokines. In a model of metabolic disruption in response to treatment with high lipids and fructose, sc-liver organoids exhibit hallmarks of steatosis and insulin resistance, while sc-islets produce pro-inflammatory cytokines on-chip. Finally, the platform reproduces known effects of anti-diabetic drugs on-chip. Taken together, the platform provides a basis for functional studies of obesity, T2DM, and MASLD on-chip, as well as for testing potential therapeutic interventions.

Correlative super-resolution analysis of cardiac calcium sparks and their molecular origins in health and disease.

Rapid release of calcium from internal stores via ryanodine receptors (RyRs) is one of the fastest types of cytoplasmic second messenger signalling in excitable cells. In the heart, rapid summation of the elementary events of calcium release, 'calcium sparks', determine the contraction of the myocardium. We adapted a correlative super-resolution microscopy protocol to correlate sub-plasmalemmal spontaneous calcium sparks in rat right ventricular myocytes with the local nanoscale RyR2 positions. This revealed a steep relationship between the integral of a calcium spark and the sum of the local RyR2s. Segmentation of recurring spark sites showed evidence of repeated and triggered saltatory activation of multiple local RyR2 clusters. In myocytes taken from failing right ventricles, RyR2 clusters themselves showed a dissipated morphology and fragmented (smaller) clusters. They also featured greater heterogeneity in both the spark properties and the relationship between the integral of the calcium spark and the local ensemble of RyR2s. While fragmented (smaller) RyR2 clusters were rarely observed directly underlying the larger sparks or the recurring spark sites, local interrogation of the channel-to-channel distances confirmed a clear link between the positions of each calcium spark and the tight, non-random clustering of the local RyR2 in both healthy and failing ventricles.

Differential labelling of human sub-cellular compartments with fluorescent dye esters and expansion microscopy.

Amine-reactive esters of aromatic fluorescent dyes are emerging as imaging probes for nondescript staining of cellular and tissue architectures. We characterised the staining patterns of 14 fluorescent dye ester species with varying physical and spectral properties in the broadly studied human HeLa cell line. When combined with the super-resolution technique expansion microscopy (ExM) involving swellable acrylamide hydrogels, fluorescent esters reveal nanoscale features including cytoplasmic membrane-bound compartments and nucleolar densities. We observe differential labelling patterns linked to the biochemical properties of the conjugated dye. Alterations in staining density and compartment specificity were seen depending on the timepoint of application in the ExM protocol. Additional complexity in labelling patterns was detected arising from inter-ester interactions. Our findings raise a number of considerations for the use of fluorescent esters. We demonstrate esters as a useful addition to the repertoire of stains of the cellular proteome, whether applied either on their own to visualise overall cellular morphology, or as counterstains providing ultrastructural context alongside specific target markers like antibodies.

Super-Resolution Analysis of the Origins of the Elementary Events of ER Calcium Release in Dorsal Root Ganglion Neurons.

Coordinated events of calcium (Ca2+) released from the endoplasmic reticulum (ER) are key second messengers in excitable cells. In pain-sensing dorsal root ganglion (DRG) neurons, these events can be observed as Ca2+ sparks, produced by a combination of ryanodine receptors (RyR) and inositol 1,4,5-triphosphate receptors (IP3R1). These microscopic signals offer the neuronal cells with a possible means of modulating the subplasmalemmal Ca2+ handling, initiating vesicular exocytosis. With super-resolution dSTORM and expansion microscopies, we visualised the nanoscale distributions of both RyR and IP3R1 that featured loosely organised clusters in the subplasmalemmal regions of cultured rat DRG somata. We adapted a novel correlative microscopy protocol to examine the nanoscale patterns of RyR and IP3R1 in the locality of each Ca2+ spark. We found that most subplasmalemmal sparks correlated with relatively small groups of RyR whilst larger sparks were often associated with larger groups of IP3R1. These data also showed spontaneous Ca2+ sparks in <30% of the subplasmalemmal cell area but consisted of both these channel species at a 3.8-5 times higher density than in nonactive regions of the cell. Taken together, these observations reveal distinct patterns and length scales of RyR and IP3R1 co-clustering at contact sites between the ER and the surface plasmalemma that encode the positions and the quantity of Ca2+ released at each Ca2+ spark.

Three-dimensional visualization of the cardiac ryanodine receptor clusters and the molecular-scale fraying of dyads.

Clusters of ryanodine receptor calcium channels (RyRs) form the primary molecular machinery of intracellular calcium signalling in cardiomyocytes. While a range of optical super-resolution microscopy techniques have revealed the nanoscale structure of these clusters, the three-dimensional (3D) nanoscale topologies of the clusters have remained mostly unresolved. In this paper, we demonstrate the exploitation of molecular-scale resolution in enhanced expansion microscopy (EExM) along with various 2D and 3D visualization strategies to observe the topological complexities, geometries and molecular sub-domains within the RyR clusters. Notably, we observed sub-domains containing RyR-binding protein junctophilin-2 (JPH2) occupying the central regions of RyR clusters in the deeper interior of the myocytes (including dyads), while the poles were typically devoid of JPH2, lending to a looser RyR arrangement. By contrast, peripheral RyR clusters exhibited variable co-clustering patterns and ratios between RyR and JPH2. EExM images of dyadic RyR clusters in right ventricular (RV) myocytes isolated from rats with monocrotaline-induced RV failure revealed hallmarks of RyR cluster fragmentation accompanied by breaches in the JPH2 sub-domains. Frayed RyR patterns observed adjacent to these constitute new evidence that the destabilization of the RyR arrays inside the JPH2 sub-domains may seed the primordial foci of dyad remodelling observed in heart failure. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

Enhanced expansion microscopy to measure nanoscale structural and biochemical remodeling in single cells.

Resolution is a key feature in microscopy which allows the visualization of the fine structure of cells. Much of the life processes within these cells depend on the three-dimensional (3D) complexity of these structures. Optical super-resolution microscopies are currently the preferred choice of molecular and cell biologists who seek to visualize the organization of specific protein species at the nanometer scale. Traditional super-resolution microscopy techniques have often been limited by sample thickness, axial resolution, specialist optical instrumentation and computationally-demanding software for assembling the images. In this chapter we detail the protocol, "enhanced expansion microscopy" (EExM), which combines X10 expansion microscopy with Airyscan confocal microscopy. EExM enables 15nm lateral (and 35nm axial) resolution, and is a relatively cheap, accessible option allowing single protein resolution for the non-specialist optical microscopists. We illustrate how EExM has been utilized for mapping the 3D topology of intracellular protein arrays at sample depths which are not always compatible with some of the traditional super-resolution techniques. We demonstrate that antibody markers can recognize and map post-translational modifications of individual proteins in addition to their 3D positions. Finally, we discuss the current uncertainties and validations in EExM which include the isotropy in gel expansion and assessment of the expansion factor (of resolution improvement).

Pre-injury estimates of subjective quality of life following traumatic brain injury.

PRIMARY OBJECTIVE: To compare the pre-injury subjective quality of life (SQOL) estimates of a representative sample of adults with TBI, using the Quality of Life Inventory (QOLI) with the measure's generic US-based norms and identify a factor structure for the instrument within the local TBI population. RESEARCH DESIGN: A population-based, cross-sectional design conducted with data collected by the Neurotrauma Register of Tasmania (2003-2005). METHODS AND PROCEDURES: As soon as possible following their emergence from post-traumatic amnesia, 470 participants provided pre-injury estimates of their SQOL using the QOLI. The distribution of this sample was compared with the measure's normative distribution. The sample was separated evenly into two groups (n = 235) for separate exploratory and confirmatory factor analyses. MAIN OUTCOMES AND RESULTS: Small differences were found between the pre-injury estimates and the QOLI's US-based normative distribution. Corrections were provided to clinical classification ranges for this population. Three factors were identified and confirmed for the QOLI in separate TBI samples. CONCLUSION: The results of this study support the use of the QOLI in measuring SQOL in TBI rehabilitation and outcomes research.

Three-Dimensional and Chemical Mapping of Intracellular Signaling Nanodomains in Health and Disease with Enhanced Expansion Microscopy.

Nanodomains are intracellular foci which transduce signals between major cellular compartments. One of the most ubiquitous signal transducers, the ryanodine receptor (RyR) calcium channel, is tightly clustered within these nanodomains. Super-resolution microscopy has previously been used to visualize RyR clusters near the cell surface. A majority of nanodomains located deeper within cells have remained unresolved due to limited imaging depths and axial resolution of these modalities. A series of enhancements made to expansion microscopy allowed individual RyRs to be resolved within planar nanodomains at the cell periphery and the curved nanodomains located deeper within the interiors of cardiomyocytes. With a resolution of ∼ 15 nm, we localized both the position of RyRs and their individual phosphorylation for the residue Ser2808. With a three-dimensional imaging protocol, we observed disturbances to the RyR arrays in the nanometer scale which accompanied right-heart failure caused by pulmonary hypertension. The disease coincided with a distinct gradient of RyR hyperphosphorylation from the edge of the nanodomain toward the center, not seen in healthy cells. This spatial profile appeared to contrast distinctly from that sustained by the cells during acute, physiological hyperphosphorylation when they were stimulated with a ß-adrenergic agonist. Simulations of RyR arrays based on the experimentally determined channel positions and phosphorylation signatures showed how the nanoscale dispersal of the RyRs during pathology diminishes its intrinsic likelihood to ignite a calcium signal. It also revealed that the natural topography of RyR phosphorylation could offset potential heterogeneity in nanodomain excitability which may arise from such RyR reorganization.

A new perspective on concepts of asthma severity and control.

Concepts of asthma severity and control are important in the evaluation of patients and their response to treatment but the terminology is not standardised and the terms are often used interchangeably. This review, arising from the work of an American Thoracic Society/European Respiratory Society Task Force, identifies the need for separate concepts of control and severity, describes their evolution in asthma guidelines and provides a framework for understanding the relationship between current concepts of asthma phenotype, severity and control. "Asthma control" refers to the extent to which the manifestations of asthma have been reduced or removed by treatment. Its assessment should incorporate the dual components of current clinical control (e.g. symptoms, reliever use and lung function) and future risk (e.g. exacerbations and lung function decline). The most clinically useful concept of asthma severity is based on the intensity of treatment required to achieve good asthma control, i.e. severity is assessed during treatment. Severe asthma is defined as the requirement for (not necessarily just prescription or use of) high-intensity treatment. Asthma severity may be influenced by the underlying disease activity and by the patient's phenotype, both of which may be further described using pathological and physiological markers. These markers can also act as surrogate measures for future risk.

Repeated infusions of levosimendan: well tolerated and improves functional capacity in decompensated heart failure - a single-centre experience.

BACKGROUND: Levosimendan is a novel agent used in the treatment of patients with decompensated heart failure to enhance cardiac contractility. Recent clinical studies have demonstrated that single doses of levosimendan have positive symptomatic and haemodynamic benefits, few have explored the efficacy and safety of intermittent repeated doses of levosimendan. AIMS: In this prospective study we document our single-centre experience of repeated administration of levosimendan to patients with decompensated heart failure. METHODS: Prospective data were collected and analysed with respect to New York Heart Association (NYHA) class, mean arterial pressure (MAP), brain natriuretic peptide levels (BNP) and adverse events. RESULTS: Forty-four consecutive patients with decompensated heart failure received repeated doses of levosimendan. The mean dosing interval was 66.2 (12) days. All patients had documented evidence of impaired left ventricular function, with a mean ejection fraction (EF) of 23.7% (2.2). Fifty-eight percent were NYHA class IV, mean age 50 (2.4), 82% were male. A significant drop in BNP levels and improvement in NYHA class was seen post-infusion. In general, levosimendan was well tolerated with 130 (83.5%) infusions completed without an adverse event. Twenty-five percent of patients were bridged to cardiac transplant or left ventricular assist device (LVAD) insertion. Four patients received 12 infusions, in total in the community. CONCLUSION: The majority of repeated levosimendan infusions were well tolerated, reduced BNP and improved NYHA functional class. In selected patients it can be administered in the community. Further investigation is required to assess the efficacy and safety of this approach.

GafChromic RTQA film for routine quality assurance of high-energy photon beams.

Self-developing film offers many advantages over conventional radiographic verification film for routine radiotherapy quality assurance (QA). This paper presents results from an initial evaluation of a beam measurement system using GafChromic RTQA film and a flatbed scanner. Variability and energy dependence of the film calibration and accuracy of scanner readout are investigated in the context of QA measurements. For exposures of film between 2 and 4 Gy, the system is adequate for measurement of beam dimensions, as in multi-leaf collimator (MLC) offsets and secondary jaw calibrations, where agreement with conventional film measurements is within 0.5 mm. However, the measurement of absolute dose is subject to errors of about 25 cGy.

Determination of the prognostic value of cyclin D1 overexpression in breast cancer.

Cyclin D1 plays a critical role in the timing of the initiation of DNA synthesis in the normal cell cycle of mammalian cells. Deregulated expression of this protein has been seen in a variety of tumours either as a result of gene amplification or chromosomal translocation, in breast cancer and B cell malignancies respectively. In order to determine the role this putative oncoprotein plays in breast cancer, we have applied a new monoclonal antibody, recently produced in our laboratory, in an immunohistochemical study of 93 primary breast carcinomas. We show that approximately 28% of the cases displayed enhanced expression of the cyclin D1 protein. Furthermore, either cyclin D1, cyclin D3, or both, were expressed in 69% of cases, suggesting that overexpression of any one member of this family may relieve cancer cells of their mitogenic stimulatory requirement. In addition, we show that those patients whose breast cancers co-express cyclin D1 with either epidermal growth factor receptor (EGFR) or the retinoblastoma protein (pRB) have a significantly poorer prognosis in comparison to those expressing cyclin D1 alone. Our observations indicate that, in a subset of breast cancers, aberrant cyclin D1 expression is a contributory factor to tumorigenesis and in association with EGFR or pRB expression, identify those tumours which may require more aggressive therapy.

Identifying the predictors of acute urinary retention following magnetic-resonance-guided prostate brachytherapy.

PURPOSE: Larger prostate gland volumes have been associated with long-term urinary morbidity in prostate interstitial radiation therapy utilizing ultrasound image guidance technique. This study was performed to identify the clinical and technical predictors of acute urinary retention following magnetic-resonance (MR)-guided prostate interstitial brachytherapy. METHODS AND MATERIALS: Fifty patients underwent MR-guided prostate brachytherapy between December 1997 and March 1999. Patient selection was limited to men with stage T1cNXM0 disease, PSA of less than10 ng/mL, biopsy Gleason score not more than 3 + 4, and endorectal coil MR stage T2 disease. Dosimetry plans were developed in the operating room and (125)Iodine sources were implanted using MR real-time guidance. The peripheral zone (PZ) of the prostate gland was defined as the clinical target volume (CTV) and the minimum prescribed dose to the CTV was 137 Gy. The volumes of the PZ, transition zone (TZ), and total prostate gland volume were also determined by MR. Individual source strength ranged from 0.35 to 0.54 microGym(2)/h (NIST 99, median 0.46 microGym(2)/h) and the total implanted activity ranged from 17.0 to 43.1 mCi (median, 28.1 mCi) using 43-120 seeds (median, 79). The seeds were placed using MR-compatible biopsy needles (14-28, median, 19). RESULTS: The ability of clinical (MR defined prostate, PZ, and TZ volumes) and technical (number of catheters, number of seeds implanted, and total activity) factors to predict AUR for 50 men undergoing MR-guided prostate interstitial brachytherapy were evaluated using univariable and logistic regression multivariable analyses. Six men (12%) experienced AUR within 24 h after removal of the Foley catheter subsequent to prostate brachytherapy. The total number of seeds (p = 0.05), MR determined prostate volume (p < 0.01), and the MR-determined TZ volume (p < 0.01) were significant predictors of AUR on univariable analysis. Utilizing a multivariable logistic regression analysis, the TZ volume was the only significant predictor of AUR (p < 0.01). The prostate volume is highly correlated to the TZ volume (Spearman correlation coefficient of 0. 91) and was thus significant in the univariable analysis; however, the prostate volume did not add prognostic value in multivariable analysis. CONCLUSION: Benign prostatic hyperplasia (BPH) resulting in an enlarged TZ volume, is the most important predictor of AUR following MR-guided prostate interstitial radiation therapy. Although AUR was significant (60%) in men with moderate BPH (TZ volume >/= 50 cc), it was also self-limiting.

Subcloning using simplified adaptor addition.

A simplified procedure for the addition of synthetic oligonucleotide adaptors to subclone DNA fragments with incompatible ends is presented. An organophosphate degradation gene on a PstI fragment was cloned into the HindIII site of the fungal vector pH1S. The opd gene specifies parathion hydrolase and was first isolated from a Flavobacterium sp. The gene was present in 12% of the plasmids recovered and was inserted in either direction with similar frequencies: 53% with the opd start codon distal to the single SalI site of pH1S and 47% in the other orientation. All enzymatic steps were carried out in a single microconcentrator eliminating DNA loss through manipulation and transfer. Normally, during adaptor or linker addition, a larger number of oligonucleotides are attached at each end of the insert DNA and must be removed before cloning. The need for enzymatic digestion to remove excess adaptors was avoided. Traditional methods have utilized phenol/chloroform extraction, ethanol precipitation, gel filtration chromatography, spermine precipitation, or preparative gel electrophoresis. Eliminating these steps resulted in a simpler, more reliable procedure.

A novel quantitative immunoassay system for p53 using antibodies selected for optimum designation of p53 status.

AIM: To develop a highly sensitive and specific enzyme linked immunosorbent assay (ELISA) system for analysis of p53 protein in cancer lysates. METHODS: The anti-p53 monoclonal antibodies DO7, 1801, BP53.12, and 421, and anti-p53 polyclonal antiserum CM1 were assessed by immunohistochemistry and western blot analysis to identify those most suitable for determining p53 status of cancer cells. Antibodies with desired characteristics were used to develop a non-competitive sandwich type ELISA system for analysis of p53 expression in cancer cytosols. Using the ELISA, p53 protein concentrations were measured in a small series of breast cancers, and the quantitative values compared with p53 immunohistochemical data of the same cancers. RESULTS: DO7 and 1801 gave the most specific and reliable results on immunohistochemistry and western blot analysis. Using these two antibodies, a non-competitive sandwich type ELISA system was developed to analyse p53 quantitatively. Analysis of the breast cancer series showed a good correlation between immunohistochemistry and the ELISA-tumours were generally positive using both techniques. Discrepancies were noted however: some cancers were immunohistochemically negative but ELISA positive. One explanation for this may be that the ELISA is more sensitive than immunohistochemistry. CONCLUSION: The p53 ELISA system is a non-competitive double monoclonal antibody sandwich method, using DO7 and 1801 which have been shown to be highly specific for p53 protein by immunohistochemistry and western blot analysis. The lower threshold of the assay is 0.1 ng/ml analyte in an enriched recombinant p53 preparation. As p53 is now regarded as a protein associated with prognosis in breast and other cancers, the assay may have clinical applications.