Neurobiomarker and body temperature responses to recreational marathon running.

OBJECTIVES: To assess how biomarkers indicating central nervous system insult (neurobiomarkers) vary in peripheral blood with exertional-heat stress from prolonged endurance exercise. DESIGN: Observational study of changes in neuron specific enolase (NSE), S100 calcium-binding protein B (S100ß), Glial Fibrillary Acid Protein (GFAP) and Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) at Brighton Marathon 2022. METHODS: In 38 marathoners with in-race core temperature (Tc) monitoring, exposure (High, Intermediate or Low) was classified by cumulative hyperthermia - calculated as area under curve of Time × Tc > 38 °C - and also by running duration (finishing time). Blood was sampled for neurobiomarkers, cortisol and fluid-regulatory stress surrogates, including copeptin and creatinine (at rested baseline; within 30 min of finishing; and at 24 h). RESULTS: Finishing in 236 ±â€¯40 min, runners showed stable GFAP and UCH-L1 across the marathon and next-day. Significant (P < 0.05) increases from baseline were shown post-marathon and at 24 h for S100ß (8.52 [3.65, 22.95] vs 39.0 [26.48, 52.33] vs 80.3 [49.1, 99.7] ng·L-1) and post-marathon only for NSE (3.73 [3.30, 4.32] vs 4.85 [4.45, 5.80] µg·L-1, P < 0.0001). Whilst differential response to hyperthermia was observed for cortisol, copeptin and creatinine, neurobiomarker responses did not vary. Post-marathon, only NSE differed by exercise duration (High vs Low, 5.81 ±â€¯1.77 vs. 4.69 ±â€¯0.73 µg·L-1, adjusted P = 0.0358). CONCLUSIONS: Successful marathon performance did not associate with evidence for substantial neuronal insult. To account for variation in neurobiomarkers with prolonged endurance exercise, factors additional to hyperthermia, such as exercise duration and intensity, should be further investigated.

Non-theatre emergency airway management: a multicentre prospective observational study.

Emergency airway management events are common, unpredictable and associated with high complication rates. This multicentre prospective observational study across eight acute NHS hospitals in southeast England reports the incidence and nature of non-theatre emergency airway management events. Data were collected from non-theatre emergency airway management, including adverse events, over a continuous 28-day window, and recorded on an electronic case report form. Events were classified according to type (advanced airway; simple airway; and cardiac arrest). A total of 166 events were recorded, with 111 advanced airway events involving tracheal intubation or tracheostomy management. Senior personnel with three or more years of airway management experience were present for 105/111 (95%) advanced airway management episodes. There was a significant reduction in consultant or equivalent presence out-of-hours (21/64, 33%) vs. in-hours (34/47, 72%) (p < 0.001). We found high utilisation of videolaryngoscopy (95/106, 90%) and universal use of capnography for all advanced airway management events. This was lower during cardiac arrest when videolaryngoscopy was used in 11/16 (69%) of tracheal intubations and capnography in 21/32 (66%) of all cardiac arrest episodes. Adverse outcomes during advanced airway management (excluding during cardiac arrest) occurred in 53/111 (48%) episodes, including hypoxia (desaturation to Sp O2 < 80% in 14/111, 13%) and hypotension (systolic blood pressure < 80 mmHg in 27/111, 25%). Adverse outcomes were not associated with time of day or experience level of airway practitioners. We conclude that there is a disparity between consultant presence for advanced airway interventions in- and out-of-hours; high utilisation of videolaryngoscopy and capnography, especially for advanced airway interventions; and a high incidence of hypotension and hypoxaemia, including critical values, during non-theatre airway management.

First steps: Parent health behaviours related to children's foot health.

Good foot health throughout childhood is important but remains poorly understood with few studies exploring this topic. The aim of this study was to define parents' knowledge, practices and health-related perceptions of children's feet. A qualitative design was adopted. Semi-structured, one-to-one interviews were carried out with parents of children aged five years and under, recruited from South East and North West of England. Interviews explored parents' views, beliefs and understanding of foot health in infancy and early childhood. Transcripts of the interviews were analysed using thematic analysis. Eighteen interviews were conducted. Seven themes were identified relating to (1) parents belief and knowledge about children's foot health; (2) how parents use and share foot health information; (3) activities for supporting foot health and development; (4) footwear choices, beliefs and influences; (5) the way they access health professionals; (6) the way they search for foot health information and (7) developing practice(s) to support parents. The study provides the first insight into how parents view foot health in early infancy and childhood. The findings highlight the key foot health beliefs important to parents, how they learn about and what influences their decision-making about caring for children's feet, the way parents receive and seek information, and how they access support for foot health concerns. The findings highlight the need for accurate, clear and consistent foot health messages, and the important role health professionals have in signposting parents towards reliable and informative sources on foot health.

Small vessel disease and intracoronary plaque composition: a single centre cross-sectional observational study.

Cardiac events are commonly triggered by rupture of intracoronary plaque. Many studies have suggested that retinal small vessel abnormalities predict cardiac events. The present study examined retinal microvascular abnormalities associated with intracoronary plaque. This was a single centre cross-sectional observational study of consecutive subjects who underwent coronary angiography and intracoronary optical coherence tomography (OCT) of occlusive coronary artery disease. Subjects' retinal images were deidentified and graded for microvascular retinopathy (Wong and Mitchell classification), and vessel calibre using a semiautomated method based on Knudtson's modification of the Parr Hubbard formula. Control subjects had no significant plaque on angiography. Analysis used the Fisher's exact test or student t-test. Thirty-two subjects with intracoronary plaque including 22 males (79%) had a mean age of 62.6 ± 9.4 years. Twenty-four (86%) had hypertension, 10 (36%) had diabetes, and 21 (75%) were current or former smokers. Their average mean arterial pressure was 90.5 ± 5.8 mm Hg, and mean eGFR was 74 ± 15/min/1.73 m2. On angiography, 23 (82%) had a left anterior descending artery (LAD) stenosis, their mean diseased vessel score was 1.86 ± 1.21, and mean total stent number was 1.04 ± 1.00. Plaque type was mainly (>50%) fibrous (n = 7), lipid (n = 7), calcific (n = 10), or mixed (n = 4). Control subjects had a lower mean diastolic BP (p = 0.01), were less likely to have an LAD stenosis (p < 0.001), a lower mean diseased vessel score (p < 0.001) and fewer stents (p = 0.02). Subjects with plaque were more likely to have a moderate microvascular retinopathy than those with none (p = 0.004). Moderate retinopathy was more common with lipid (p = 0.05) or calcific (p = 0.003) plaque. Individuals with calcific plaque had a larger arteriole calibre (158.4 ± 15.2 µm) than those with no plaque (143.8 ± 10.6 µm, p = 0.02), but calibre was not related to diabetes or smoking. Calibre did not correlate with plaque length, thickness or arc angle. Thus, subjects with intracoronary artery plaque are more likely to have a moderate microvascular retinopathy. Those with calcific plaque have larger retinal arterioles which is consistent with our previous finding of larger vessel calibre in triple coronary artery disease. Retinal microvascular imaging warrants further evaluation in identifying severe coronary artery disease.

Acute kidney injury associated with endurance events-is it a cause for concern? A systematic review.

INTRODUCTION: A growing body of evidence suggests even small rises in serum creatinine (SCr) are of considerable clinical relevance. Given that participants in endurance events are exposed to potential (repeated) renal insults, a systematic review was undertaken to collate current evidence for acute kidney injury (AKI), complicating such events. METHODS: A systematic review of studies and case reports meeting inclusion criteria on Medline and EMBASE (inception to October 2015). Included: studies with markers of renal function before and after endurance or ultraendurance events; case reports of severe AKI. Two reviewers assessed risk of bias using the Newcastle-Ottawa scale. RESULTS: Eleven case report publications (n=27 individuals) of severe AKI, were retrieved, with risk factors including systemic illness or nephrotoxic medications usually identified. From 30 studies of endurance and ultraendurance events, mean rise in SCr was 29 (±12.3) µmol/L after marathon or ultramarathon (17 studies, n=568 participants) events. Where follow-up tests were conducted, SCr returned to baseline within 48 hours. Rises in biomarkers suggest potential parenchymal insult, rather than simply muscle breakdown. However, evidence of long-term deleterious effects is lacking. CONCLUSIONS: Raised levels of SCr are reported immediately after endurance events. It is not clear whether this is either clinically significant, or if repeated participation predisposes to long-term sequelae. The aetiology of severe exercise-associated AKI is usually multifactorial, with risk factors generally identified in the rare cases reported. On-site biochemistry, urine analysis and biomarkers of AKI may help identify collapsed runners who are at significant short-term risk and allow suitable follow-up.

Predicting AKI in emergency admissions: an external validation study of the acute kidney injury prediction score (APS).

OBJECTIVES: Hospital-acquired acute kidney injury (HA-AKI) is associated with a high risk of mortality. Prediction models or rules may identify those most at risk of HA-AKI. This study externally validated one of the few clinical prediction rules (CPRs) derived in a general medicine cohort using clinical information and data from an acute hospitals electronic system on admission: the acute kidney injury prediction score (APS). DESIGN, SETTING AND PARTICIPANTS: External validation in a single UK non-specialist acute hospital (2013-2015, 12 554 episodes); four cohorts: adult medical and general surgical populations, with and without a known preadmission baseline serum creatinine (SCr). METHODS: Performance assessed by discrimination using area under the receiver operating characteristic curves (AUCROC) and calibration. RESULTS: HA-AKI incidence within 7 days (kidney disease: improving global outcomes (KDIGO) change in SCr) was 8.1% (n=409) of medical patients with known baseline SCr, 6.6% (n=141) in those without a baseline, 4.9% (n=204) in surgical patients with baseline and 4% (n=49) in those without. Across the four cohorts AUCROC were: medical with known baseline 0.65 (95% CIs 0.62 to 0.67) and no baseline 0.71 (0.67 to 0.75), surgical with baseline 0.66 (0.62 to 0.70) and no baseline 0.68 (0.58 to 0.75). For calibration, in medicine and surgical cohorts with baseline SCr, Hosmer-Lemeshow p values were non-significant, suggesting acceptable calibration. In the medical cohort, at a cut-off of five points on the APS to predict HA-AKI, positive predictive value was 16% (13-18%) and negative predictive value 94% (93-94%). Of medical patients with HA-AKI, those with an APS ≥5 had a significantly increased risk of death (28% vs 18%, OR 1.8 (95% CI 1.1 to 2.9), p=0.015). CONCLUSIONS: On external validation the APS on admission shows moderate discrimination and acceptable calibration to predict HA-AKI and may be useful as a severity marker when HA-AKI occurs. Harnessing linked data from primary care may be one way to achieve more accurate risk prediction.

Macrophage contact induces RhoA GTPase signaling to trigger tumor cell intravasation.

Most cancer patients die as a result of metastasis, thus it is important to understand the molecular mechanisms of dissemination, including intra- and extravasation. Although the mechanisms of extravasation have been vastly studied in vitro and in vivo, the process of intravasation is still unclear. Furthermore, how cells in the tumor microenvironment facilitate tumor cell intravasation is still unknown. Using high-resolution imaging, we found that macrophages enhance tumor cell intravasation upon physical contact. Macrophage and tumor cell contact induce RhoA activity in tumor cells, triggering the formation of actin-rich degradative protrusions called invadopodia, enabling tumor cells to degrade and break through matrix barriers during tumor cell transendothelial migration. Interestingly, we show that macrophage-induced invadopodium formation and tumor cell intravasation also occur in patient-derived tumor cells and in vivo models, revealing a conserved mechanism of tumor cell intravasation. Our results illustrate a novel heterotypic cell contact-mediated signaling role for RhoA, as well as yield mechanistic insight into the ability of cells within the tumor microenvironment to facilitate steps of the metastatic cascade.

Feedback of personal retinal images appears to have a motivational impact in people with non-proliferative diabetic retinopathy and suboptimal HbA1c: findings of a pilot study.

AIM: To conduct a pilot study to explore the potential impact of visual feedback of personal retinal images on diabetes outcomes. METHODS: Twenty-five participants with non-proliferative diabetic retinopathy and suboptimal HbA(1c) (> 53 mmol/mol; > 7%) were randomized to receive visual feedback of their own retinal images or to a control group. At baseline and 3-month follow-up, HbA(1c), standard measures of beliefs, diabetes-related distress and self-care activities were assessed. RESULTS: In unadjusted models, relative to controls, the intervention group showed significantly greater improvement in HbA(1c) at 3-month follow-up (-0.6% vs. +0.3%, P < 0.01), as well as enhanced motivation to improve blood glucose management (P < 0.05). CONCLUSIONS: This small pilot study provides preliminary evidence that visual feedback of personal retinal images may offer a practical educational strategy for clinicians in eye care services to improve diabetes outcomes in non-target compliant patients. A fully powered randomized controlled trial is required to confirm these findings and determine the optimal use of feedback to produce sustained effects.

Problem based review: pleuritic chest pain.

Pleuritic pain, a sharp discomfort near the chest wall exacerbated by inspiration is associated with a number of pathologies. Pulmonary embolus and infection are two common causes but diagnosis can often be challenging, both for experienced physicians and trainees. The underlying anatomy and pathophysiology of such pain and the most common aetiologies are presented. Clinical symptoms and signs that may arise alongside pleuritic pain are then discussed, followed by an introduction to the diagnostic tools such as the Wells’ score and current guidelines that can help to select the most appropriate investigation(s). Management of pulmonary embolism and other common causes of pleuritic pain are also discussed and highlighted by a clinical vignette.

OS010. Use of retinal imaging to characterise physiological vascularchanges throughout pregnancy.

INTRODUCTION: Visualisation of the microcirculation through retinal imaging can provide information on the health of systemic vasculature. Characterisation of the retinal vasculature throughout pregnancy using retinal imaging is a novel approach to examine physiological changes to the cardiovascular system, and may be useful to predict early pathophysiological signs of adverse maternal outcomes. OBJECTIVES: To characterise the retinal vascular and blood pressure (BP) changes that occur throughout a healthy pregnancy. METHODS: Data was collected from women recruited at 13±2 weeks of gestation from Royal Prince Alfred Hospital, a major tertiary referral hospital in Sydney, Australia. Retinal images centred on the optic disc and BP readings were collected throughout pregnancy. Postnatal data was collected from medical records, and women with hypertensive disorders of pregnancy and gestational diabetes mellitus were excluded. This left a final group of 19 women. Retinal images from 13±2, 19±2, 29±2 and 38±2 weeks gestation were graded using semi-automated retinal vascular calibre measurement (IVAN) software and the central retinal arteriolar equivalent (CRAE), and central retinal venular equivalent (CRVE). BP data was collected at the same time points as the retinal images. Analysis of data was performed using paired t-tests and repeated measures analysis of variance (ANOVA). Women with missing data points were excluded from the analysis at the relevant time points. RESULTS: Over the course of pregnancy, there was a significant dilatation of retinal arterioles between 13±2 and 19±2weeks (from 166.4 to 172.7µm, SE: 3.7µm, n=19, p=0.01), corresponding to a significant fall in diastolic BP during this time (from 64.6 to 60.2mmHg, SE: 1.5mmHg, p=0.01). No significant changes in venular diameter or systolic BP were noted. Between 19±2 and 29±2weeks (n=4), no significant changes to retinal arteriolar or venular diameter were seen although there were significant increases in both systolic and diastolic BP (SBP: from 100.3 to 109.9mmHg, SE: 1.9mmHg, p=0.01; DBP: from 59.3 to 64.6mmHg, SE: 6.9mmHg, p=0.01). Between 29±2 and 38±2weeks (n=3), no significant changes in retinal arteriolar, and venular diameter or BP were observed. CONCLUSION: An increase in retinal arteriolar diameter between 13±2 and 19±2 weeks gestation was observed, which corresponded to a decrease in both systolic and diastolic BP. However, between 19±2 and 29±2 weeks there was no change in vasculature, even though there was a significant increase in BP. By characterising the changes to retinal vessels that occur throughout a healthy pregnancy, we can further our understanding of the response of the systemic vasculature to pregnancy, which may provide clues to early vascular disease of pregnancies.

OS016. Retinal vascular changes in hypertensive disorders of pregnancy.

INTRODUCTION: Hypertensive disorders of pregnancy (HDP) are characterised by vascular dysfunction. Retinal vascular imaging is a novel, non-invasive way to characterise early microvascular changes in pregnancy, and as a result has the potential to be used to predict the onset of HDP. OBJECTIVES: To characterise retinal vascular changes that occur in HDP, and compare these changes to those in healthy pregnancies. METHODS: Women were recruited at 13±2 weeks of gestation from Royal Prince Alfred Hospital, a major metropolitan tertiary referral hospital in Sydney, Australia. Retinal images centred on the optic disc and blood pressure (BP) readings were collected at 13±2, 19±2, 29±2 and 38±2 weeks gestation. Retinal images were graded using semi-automated retinal vascular calibre measurement software (IVAN) and the central retinal arteriolar equivalent (CRAE) and central retinal venular equivalent (CRVE) were calculated. Within and between subject repeat measures analysis was performed on images from each trimester, using paired t-tests and repeated measures analysis of variance (ANOVA). Multiple linear regressions were used to model the average arteriole diameter adjusted for age, tobacco consumption and body mass index (BMI). All tests were two-sided using a 5% level of significance. A clinical diagnosis of HDP was obtained from postnatal medical record data. Women with missing data points were excluded from the analysis at that time point. RESULTS: Of the 39 women included in the study, 6 (15%) were diagnosed with HDP. In the HDP cohort, repeated measures ANOVA revealed no significant changes in arteriolar or venular diameter measurements throughout pregnancy. Paired t-tests indicated no significant differences in any of the outcome measures between HDP and healthy pregnancies at 13±2 (n=36) and 19±2 (n=39)weeks. At 29±2weeks (n=39), there was a significantly smaller venular diameter in HDP pregnancies (220.4±6.9µm vs 239.1± 5.4µm in healthy pregnancies, p=0.03). At 38±2weeks (n=39), arteriolar diameter was significantly smaller in HDP pregnancies (148.6±6.0µm vs 164.1±4.6µm in healthy pregnancies, p=0.04). Similar results persisted following adjustments for cardiovascular risk factors (age, tobacco use and BMI). CONCLUSION: Significant differences in the retinal vasculature develop in HDP as compared to healthy pregnancies. These differences appear at29±2weeks gestation and persist throughout the rest of the pregnancy. Retinal vascular imaging is a promising tool for the detection of the early microvascular changes in HDP, prior to diagnosis.

Retinal vessel calibre and micro- and macrovascular complications in type 1 diabetes.

AIMS/HYPOTHESIS: The purpose of the study was to evaluate the association between retinal vascular calibre and micro- and macrovascular complications in a population-based cohort of Danish type 1 diabetic patients. METHODS: This was a cross-sectional study of 208 long-surviving type 1 diabetic patients from a population-based Danish cohort. Retinal photographs were obtained at a clinical examination attended by each participant in 2007-2008, and retinal vascular calibre was measured and summarised as the central retinal artery or vein equivalent (CRAE or CRVE) using a computer-based program and a standardised protocol. Associations between retinal vascular calibre and micro- and macrovascular complications were examined after adjusting for confounding clinical characteristics. RESULTS: Retinal photographs were gradable for 188 of 208 patients (90.3%). The median age and duration of diabetes for patients with gradable photos were 57.9 and 42 years, respectively. After multivariate adjustments, individuals with narrower retinal arterioles were more likely to have nephropathy (OR 2.17, 95% CI 1.29-3.68, per SD decrease in CRAE) and macrovascular disease (OR 3.17, 95% CI 1.59-6.34, per SD decrease in CRAE), but not neuropathy (OR 1.10, 95% CI 0.70-1.71, per SD decrease in CRAE). Retinal venular calibre was not associated with any micro- or macrovascular complications. CONCLUSIONS/INTERPRETATION: In type 1 diabetic patients, retinal arteriolar narrowing is associated with nephropathy and macrovascular disease independently of other clinical characteristics. If supported by further prospective studies, measurement of retinal vessel diameter may allow a non-invasive evaluation of the risk of diabetes-related complications.

Epidemiological studies of injuries in rugby league: suggestions for definitions, data collection and reporting methods.

Studies on rugby league injuries use a variety of definitions and methodologies. Consequently, comparisons of published studies are difficult. Researchers with an interest in understanding the epidemiology of rugby league injury participated in a majority agreement process. This paper provides suggestions for the definitions, data collection and reporting methods for future studies of rugby league injuries. The proposed methods and definitions were developed through the use of a majority agreement process on draft versions by all authors. Recommended definitions for injury incidence, recurrence, severity and match exposure are provided as well as injury site, type, diagnosis and causation. Suggestions for match and training injury incidence calculations are also provided for the purposes of comparison. This paper provides standard definitions that, if utilised, will enable meaningful comparison of future rugby league injury surveillance data from different countries and playing levels.

[Ca2+]i as a potential downregulator of alpha2beta1-integrin-mediated A2058 tumor cell migration to type IV collagen.

We have investigated cellular Ca2+ regulation during A2058 human melanoma cell chemotaxis to type IV collagen (CIV). We have identified alpha2beta1-integrin as the primary mediator of A2058 cell response to CIV in vitro. Integrin ligation initiated a characteristic intracellular Ca2+ concentration ([Ca2+]i) response consisting of an internal release and a receptor-mediated Ca2+ entry. Thapsigargin (TG) pretreatment drained overlapping and CIV-inducible internal Ca2+ stores while initiating a store-operated Ca2+ release (SOCR). CIV-mediated Ca2+ entry was additive to TG-SOCR, suggesting an independent signaling mechanism. Similarly, ionophore application in a basal medium containing Ca2+ initiated a sustained influx. Elevated [Ca2+]i from TG-SOCR or ionophore significantly attenuated cell migration to CIV by recruiting the Ca2+/calcineurin-mediated signaling pathway. Furthermore, low [Ca2+]i induced by EGTA application in the presence of ionophore fully restored cell motility to CIV. Together, these results suggest that [Ca2+]i signaling accompanying A2058 cell response to alpha2beta1-integrin ligation is neither necessary nor sufficient and that elevated [Ca2+]i downregulates cell motility via a calcineurin-mediated mechanism in A2058 cell chemotaxis to CIV.

Correlates of personal concerns about developing Alzheimer's disease among middle-aged persons.

We examine correlates of personal concerns about developing Alzheimer's disease (AD) among (1) adult children, 40 to 60 years of age, who have a living parent with a diagnosis of probable AD (N = 108), and (2) a matched comparison group of persons with no parental history of AD (N = 150). Using stepwise regression, predictors measuring subjective perceptions of memory functioning, overall family history of AD, knowledge of AD, and sociodemographic characteristics were entered into models for the total sample and each of the subsamples. The results indicate that worries about memory functioning play a consistent role in personal concerns about developing AD across both groups, but that additional pathways to personal concerns differ among individuals having and not having a parent with AD.

Use of green fluorescent protein-conjugated beta-actin as a novel molecular marker for in vitro tumor cell chemotaxis assay.

To study the dynamics of actin cytoskeleton rearrangement in living cells, an eukaryotic expression vector expressing a beta-actin-GFP fusion protein was generated. The expression construct when transfected into NIH3T3 fibroblast, A2058 human melanoma and 293T human embryonic kidney carcinoma cell lines expressed beta-actin-GFP fusion protein, which colocalized with endogenous cellular actin as determined by histoimmunofluorescence staining. The beta-actin-GFP was also observed to be reorganized in response to treatments with the chemoattractant type IV collagen. Cells extended pseudopodial protrusions and altered the morphology of their cortical structure in response to type IV collagen stimulation. More importantly, beta-actin-GFP accumulated in areas undergoing these dynamic cytoskeleton changes, indicating that beta-actin-GFP could participate in actin polymerization. Although ectopic expression of beta-actin-GFP lead to minor side effects on cell proliferation, these studies suggest that this strategy provides an alternative to the invasive techniques currently used to study actin dynamics and permits real-time visualization of actin rearrangements in response to environmental cues.

Extracellular lipid-mediated signaling in tumor-cell activation and pseudopod protrusion.

We have pioneered an in vitro pseudopod-generation model wherein suspended tumor cells are stimulated to form pseudopods into glass micropipettes in response to soluble collagen type IV (CIV). Pertussis toxin and removing intracellular calcium were found previously to be inhibitory to that process. We now extend those observations to dissect the roles of transmembrane calcium influx and circulating fatty acids on pseudopod extension. Removal of fatty acids from BSA in basal media resulted in abrogation of pseudopod formation, while reconstitution of free fatty acids restored cell pseudopod protrusion. We thus hypothesized that fatty acids may provide necessary pseudopod stimulatory signals. Addition of lysophosphatidic acid (LPA) to the fatty acid-free CIV solution or in an opposite pipette without CIV permitted approximately 50% pseudopod recovery in all pipette directions in a dose-dependent fashion. Thapsigargin (TG), an agent that releases internal calcium stores and causes opening of store-operated calcium channels, restored pseudopod protrusion up to 80% in CIV with fatty acid-free albumin. [Ca(2+)](i) release was non-additive when cells were stimulated by TG and LPA, suggesting overlapping [Ca(2+)](i) stores. The combination of TG and LPA in fatty acid-free albumin fully restored the pseudopod response to CIV. Addition of EGTA to chelate stimulatory media calcium blocked the pseudopod response to CIV in the presence of fatty acids. This indicates that pseudopod protrusion requires transmembrane calcium entry. Thus, extracellular lipids and calcium mobilization are required to complement CIV in pseudopod protrusion from suspended cells.