British Society of Haematology, Slide Session presented at the Annual Scientific Meeting, Brighton 2009

P>Seven cases were discussed by an expert panel at the 2009 Annual Scientific Meeting of the British Society of Haematology. These cases are presented in a similar format to that adopted for the meeting. There was an initial discussion of the presenting morphology, generation of differential diagnoses and then, following display of further presenting and diagnostic information, each case was concluded with provision of a final diagnosis.

The Role of Micronutrients in Heart Failure

Heart failure is a common condition in the Western world, particularly among elderly persons and with an ever-aging population, the incidence is expected to increase. Diet in the setting of heart failure is important--patients with this condition are advised to consume a low-salt diet and monitor their weight closely. Nutritional status of patients with heart failure also is important--those with poor nutritional status tend to have a poor long-term prognosis. A growing body of evidence suggests an association between heart failure and micronutrient status. Reversible heart failure has been described as a consequence of severe thiamine and selenium deficiency. However, contemporary studies suggest that a more subtle relationship may exist between micronutrients and heart failure. This article reviews the existing literature linking heart failure and micronutrients, examining studies that investigated micronutrient intake, micronutrient status, and the effect of micronutrient supplementation in patients with heart failure, and focusing particularly on vitamin A, vitamin C, vitamin E, thiamine, other B vitamins, vitamin D, selenium, zinc, and copper.

Starting slow: the effects of response-switching frequency on patterns of cardiovascular reactivity

Research findings suggest that switching between competing response sets can be resource demanding. The current study focused on concurrent health-relevant physiological effects of task switching by assessing cardiovascular response at varying levels of switch frequency. The participants performed a response-switching task at three different levels of response set switching frequency (low, medium and high) while measurements of blood pressure and heart rate were taken. One group was exposed to response-switching frequency conditions in the order low → medium → high, while the other group was exposed to the same task conditions in the reverse order (i.e. high → medium → low). The results showed that the participants in the low → medium → high switch frequency group recovered faster from initially heightened systolic blood pressure when compared with participants in the high → medium → low group. It is concluded that the results point to a physiological "carry over" effect associated with beginning a task at rapid response switching frequency levels, and suggest the importance of habituation to task demands as a means of offsetting potentially unhealthy levels of reactivity. Implications for modern work environments are discussed.

Reliability of blood pressure determination with the Finapres with altered physiological states or pharmacodynamic conditions

The blood pressure waveform is modified on distal propagation by phenomena such as dispersion, reflection and the state of the arterial compliance. The consequent effects are amplification and narrowing of the wave, with an increased systolic, reduced diastolic and essentially unaltered mean blood pressure. The Finapres measures the peripheral pressure using the volume clamp principle; it has not been validated under altered physiological conditions and during pharmacodynamic interventions. We studied simultaneous Finapres and brachial blood pressures (using a conventional oscillometric sphygmomanometer—Vitalmap) in ten normal volunteers at rest, and during dynamic exercise and a cold pressor test. The effects of pharmacodynamic intervention were examined following beta-adrenoceptor blockade with propranolol (160 mg) or beta-adrenoceptor modulation with the beta-adrenoceptor partial agonist celiprolol (400 mg). The Finapres systolic pressure was significantly higher than the brachial value during all three test states. The difference between the systolic pressures measured by the two devices was shown to increase significantly during the cold pressor test, but not during dynamic (supine bicycle) exercise. The Finapres diastolic pressure was significantly higher than the Vitalmap value during exercise and the cold pressor test. The differences between the two methods increased significantly over time. Beta-adrenergic blockade with propranolol or modulation with celiprolol had no significant interaction with the pressure differences between the Finapres and Vitalmap techniques. The results would support the view that the Finapres can provide blood pressure information which is robust under most circumstances. Although this pharmacodynamic intervention did not alter the relationship between the peripheral and central blood pressure, it is important to note that this dynamic relationship is sensitive to circulatory loading conditions and wave transmission characteristics; it is possible that the Finapres could be less reliable in clinical settings where potent vasoactive agents were being administered.

Phenotype-Based Discovery of 2-[(E)-2-(Quinolin-2-yl)vinyl]phenol as a Novel Regulator of Ocular Angiogenesis

Retinal angiogenesis is tightly regulated to meet oxygenation and nutritional requirements. In diseases such as proliferative diabetic retinopathy and neovascular age-related macular degeneration, uncontrolled angiogenesis can lead to blindness. Our goal is to better understand the molecular processes controlling retinal angiogenesis and discover novel drugs that inhibit retinal neovascularization. Phenotype-based chemical screens were performed using the ChemBridge Diverset^TM library and inhibition of hyaloid vessel angiogenesis in Tg(fli1:EGFP) zebrafish. 2-[(E)-2-(Quinolin-2-yl)vinyl]phenol, (quininib) robustly inhibits developmental angiogenesis at 4–10 μM in zebrafish and significantly inhibits angiogenic tubule formation in HMEC-1 cells, angiogenic sprouting in aortic ring explants, and retinal revascularization in oxygen-induced retinopathy mice. Quininib is well tolerated in zebrafish, human cell lines, and murine eyes. Profiling screens of 153 angiogenic and inflammatory targets revealed that quininib does not directly target VEGF receptors but antagonizes cysteinyl leukotriene receptors 1 and 2 (CysLT_1–2) at micromolar IC₅₀ values. In summary, quininib is a novel anti-angiogenic small-molecule CysLT receptor antagonist. Quininib inhibits angiogenesis in a range of cell and tissue systems, revealing novel physiological roles for CysLT signaling. Quininib has potential as a novel therapeutic agent to treat ocular neovascular pathologies and may complement current anti-VEGF biological agents.

Degradation of poly-L-lactide. Part 1: in vitro and in vivo physiological temperature degradation

Poly-L-Lactide is a bioresorbable polymer which degrades through hydrolysis of its ester linkage influenced by initial molecular weight and degree of crystallinity. Polymers belonging to the aliphatic polyester family currently represent the most attractive group of polymers that meet the medical and physical demands for safe clinical applications. Compression moulded PLLA pellets were produced as rods, sterilized and degraded both in vitro and in vivo (sub-dermal implantation model). The material molecular weight, crystallinity, mechanical strength and thermal properties were evaluated. In both in vitro and in vivo environments, degradation proceeded at the same rate and followed the general sequence of aliphatic polyester degradation, ruling out enzymes accelerating the degradation rate in vivo. By 44 weeks duration of implantation the PLLA rods were still biocompatible, before any mass loss was observed.

Effects of the novel (Pro(3))GIP antagonist and exendin(9-39)amide on GIP- and GLP-1-induced cyclic AMP generation, insulin secretion and postprandial insulin release in obese diabetic (ob/ob) mice: evidence that GIP is the major physiological incretin

AIMS/HYPOTHESIS: This study examined the biological effects of the GIP receptor antagonist, (Pro3)GIP and the GLP-1 receptor antagonist, exendin(9-39)amide.

METHODS: Cyclic AMP production was assessed in Chinese hamster lung fibroblasts transfected with human GIP or GLP-1 receptors, respectively. In vitro insulin release studies were assessed in BRIN-BD11 cells while in vivo insulinotropic and glycaemic responses were measured in obese diabetic ( ob/ ob) mice.

RESULTS: In GIP receptor-transfected fibroblasts, (Pro(3))GIP or exendin(9-39)amide inhibited GIP-stimulated cyclic AMP production with maximal inhibition of 70.0+/-3.5% and 73.5+/-3.2% at 10(-6) mol/l, respectively. In GLP-1 receptor-transfected fibroblasts, exendin(9-39)amide inhibited GLP-1-stimulated cyclic AMP production with maximal inhibition of 60+/-0.7% at 10(-6) mol/l, whereas (Pro(3))GIP had no effect. (Pro(3))GIP specifically inhibited GIP-stimulated insulin release (86%; p<0.001) from clonal BRIN-BD11 cells, but had no effect on GLP-1-stimulated insulin release. In contrast, exendin(9-39)amide inhibited both GIP and GLP-1-stimulated insulin release (57% and 44%, respectively; p<0.001). Administration of (Pro(3))GIP, exendin(9-39)amide or a combination of both peptides (25 nmol/kg body weight, i.p.) to fasted (ob/ob) mice decreased the plasma insulin responses by 42%, 54% and 49%, respectively (p<0.01 to p<0.001). The hyperinsulinaemia of non-fasted (ob/ob) mice was decreased by 19%, 27% and 18% (p<0.05 to p<0.01) by injection of (Pro3)GIP, exendin(9-39)amide or combined peptides but accompanying changes of plasma glucose were small.

CONCLUSIONS/INTERPRETATION: These data show that (Pro(3))GIP is a specific GIP receptor antagonist. Furthermore, feeding studies in one commonly used animal model of obesity and diabetes, (ob/ob) mice, suggest that GIP is the major physiological component of the enteroinsular axis, contributing approximately 80% to incretin-induced insulin release.

Terminal nerve-derived neuropeptide Y modulates physiological responses in the olfactory epithelium of hungry axolotls (Ambystoma mexicanum).

The vertebrate brain actively regulates incoming sensory information, effectively filtering input and focusing attention toward environmental stimuli that are most relevant to the animal's behavioral context or physiological state. Such centrifugal modulation has been shown to play an important role in processing in the retina and cochlea, but has received relatively little attention in olfaction. The terminal nerve, a cranial nerve that extends underneath the lamina propria surrounding the olfactory epithelium, displays anatomical and neurochemical characteristics that suggest that it modulates activity in the olfactory epithelium. Using immunocytochemical techniques, we demonstrate that neuropeptide Y (NPY) is abundantly present in the terminal nerve in the axolotl (Ambystoma mexicanum), an aquatic salamander. Because NPY plays an important role in regulating appetite and hunger in many vertebrates, we investigated the possibility that NPY modulates activity in the olfactory epithelium in relation to the animal's hunger level. We therefore characterized the full-length NPY gene from axolotls to enable synthesis of authentic axolotl NPY for use in electrophysiological experiments. We find that axolotl NPY modulates olfactory epithelial responses evoked by L-glutamic acid, a food-related odorant, but only in hungry animals. Similarly, whole-cell patch-clamp recordings demonstrate that bath application of axolotl NPY enhances the magnitude of a tetrodotoxin-sensitive inward current, but only in hungry animals. These results suggest that expression or activity of NPY receptors in the olfactory epithelium may change with hunger level, and that terminal nerve-derived peptides modulate activity in the olfactory epithelium in response to an animal's changing behavioral and physiological circumstances.

Endostatin modulates VEGF-mediated barrier dysfunction in the retinal microvascular endothelium

Recent evidence indicates that the anti-angiogenic peptide endostatin may modulate some of the vasomodulatory effects of vascular endothelial growth factor (VEGF) in the retina, including reduction of blood retinal barrier function although it remains uncertain how endostatin promotes endothelial barrier properties. The current study has sought to examine how physiological levels of endostatin alters VEGF-induced inner BRB function using an in vitro model system and evaluation of occludin and ZO-1 regulatory responses. In addition, the ability of exogenous endostatin to regulate VEGF-mediated retinal vascular permeability in vivo was investigated.

Retinal microvascular endothelial cells (RMEC's) were exposed to various concentrations of endostatin. In parallel studies, RMEC monolayers were treated with vascular endothelial growth factor (VEGF165). Vasopermeability of RMEC monolayers and occludin expression were determined.

Blood retinal barrier integrity was quantified in mouse retina using Evans Blue assay following intravitreal delivery of VEGF165, endostatin or a VEGF/endostatin combination.

Endostatin increased the levels of expression of occludin whilst causing no significant change in FITC-dextran flux across the RMEC monolayer. Endostatin reversed the effects of VEGF165-enhanced permeability between microvascular endothelial cells and induced phosphorylation of occludin. Evans Blue leakage from retinas treated with VEGF was 2.0 fold higher than that of contra-lateral untreated eyes (P<0.05) while leakage of eyes from endostatin treated animals was unchanged. When eyes were injected with a combination of VEGF165 and endostatin there was a significant reduction in retinal vasopermeability when compared to VEGF-injected eyes (P<0.05).

We conclude that endostatin can promote integrity of the retinal endothelial barrier, possibly by preventing VEGF-mediated alteration of tight junction integrity. This suggests that endostatin may be of clinical benefit in ocular disorders where significant retinal vasopermeability changes are present.

Expression of vascular endothelial growth factor (VEGF) and its receptors is regulated in eyes with intra-ocular tumours

The immunolocalization and gene expression of vascular endothelial growth factor (VEGF) and its cognate tyrosine kinase receptors, Flt-1 and KDR, has been studied in ocular melanomas and retinoblastomas using in situ hybridization and immunohistochemistry. Tumour-related alterations in VEGF/VEGF-receptor expression have also been examined in separate and uninvolved iris, retina and choroid of the same eyes. Although VEGF immunoreactivity in the normal retina was virtually absent, low-level VEGF expression was evident in the ganglion cell-bodies, Müller cells and in a distinct population of amacrine cells. VEGF gene expression was absent in the iris and choroid of normal eyes. In tumour-bearing eyes, high levels of VEGF protein and gene expression were observed within the vascularized regions of the tumours, while the adjacent retina and choroid showed increased VEGF levels when compared with normals. Flt-1 and KDR gene expression and immunolocalization occurred in VEGF-expressing ganglion, Müller and amacrine cells in normal eyes. Within the intra-ocular tumours, VEGF-receptor gene expression and protein was evident in the endothelial cells and also in cells close to the vessels, while in the adjacent retina, Flt-1 and KDR levels were elevated over normal, especially in the blood vessels. Flt-1 and KDR were both observed at elevated levels in the choroid and iris blood vessels. This study suggests that VEGF, Flt-1 and KDR are expressed by neural, glial and vascular elements within normal human retina. Intra-ocular tumours demonstrate a high level of VEGF and VEGF-receptor expression; within uninvolved, spatially separate retina, choroid and iris in the same eyes, expression is also elevated, especially within the vasculature. Retinal vascular endothelia may respond to high intra-ocular levels of VEGF by increasing expression of their VEGF receptors, a phenomenon which could have relevance to neoplasm-related ocular neovascularization.