Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins.

The majority of severe visual loss in the United States results from complications associated with retinal neovascularization in patients with ischemic ocular diseases such as diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity. Intraocular expression of the angiogenic protein vascular endothelial growth factor (VEGF) is closely correlated with neovascularization in these human disorders and with ischemia-induced retinal neovascularization in mice. In this study, we evaluated whether in vivo inhibition of VEGF action could suppress retinal neovascularization in a murine model of ischemic retinopathy. VEGF-neutralizing chimeric proteins were constructed by joining the extracellular domain of either human (Flt) or mouse (Flk) high-affinity VEGF receptors with IgG. Control chimeric proteins that did not bind VEGF were also used. VEGF-receptor chimeric proteins eliminated in vitro retinal endothelial cell growth stimulation by either VEGF (P < 0.006) or hypoxic conditioned medium (P < 0.005) without affecting growth under nonstimulated conditions. Control proteins had no effect. To assess in vivo response, animals with bilateral retinal ischemia received intravitreal injections of VEGF antagonist in one eye and control protein in the contralateral eye. Retinal neovascularization was quantitated histologically by a masked protocol. Retinal neovascularization in the eye injected with human Flt or murine Flk chimeric protein was reduced in 100% (25/25; P < 0.0001) and 95% (21/22; P < 0.0001) 0.0001) of animals, respectively, compared to the control treated eye. This response was evident after only a single intravitreal injection and was dose dependent with suppression of neovascularization noted after total delivery of 200 ng of protein (P < 0.002). Reduction of histologically evident neovascular nuclei per 6-microns section averaged 47% +/- 4% (P < 0.001) and 37% +/- 2% (P < 0.001) for Flt and Flk chimeric proteins with maximal inhibitory effects of 77% and 66%, respectively. No retinal toxicity was observed by light microscopy. These data demonstrate VEGF's causal role in retinal angiogenesis and prove the potential of VEGF inhibition as a specific therapy for ischemic retinal disease.

Description of the fossil manganese nodules found in the Oligocene clay deposits of Moravia, Brno area, Czech Republic

In the green Oligocene clay of Krizanovice (former Krzyzanowicz) the author found numerous black nodules. In the 3-4 centimeter thick black crust of a particular specimen the concentration in MnO2 is evaluated at 46.6% MnO2. The determination was done using the Volhard's method (precipitation of Fe by ZnO and titration with KMnO4). Only the dissoleved part in HCL was analysed. The non soluble residue was essentially a silica precipitate in the form of many gray flakes. The specific gravity of the crust was evaluated at 3.8. In the internal yellow core the amount of manganese is about 2.39% MnO2. Due to the light color it is judged to probably be in the form of Mn2O3.

Chemical composition of the soluble fraction of oxidate portions of Lakes Shebadnowan ferromanganese concretions

Shebandowan Lakes, Ontario, are the site of at least 49 shallow (2-12 m) ferromanganese concretion deposits, widely distributed throughout the 48 km of the watercourse. X-ray diffraction and Mossbauer methods have revealed the presence of goethite in some of the concretions. Chemical analyses of the acid soluble portions of 72 samples gave an average composition of 43.1% Fe and 5.65% Mn with a low content of trace elements. The Shebandowan concretions are among the richest in iron of lake concretions reported, possibly because only the acid soluble portion was analysed. Their low content of trace elements suggests rapid growth rates and a relatively young age. A positive correlation was found between Mn and K, Ca, Mg, Cu, Ni, and Co and the relationship between the last three and Mn was deemed significant. Zn was independent of association with either Mn and Fe, probably due to the presence locally of zinc sulphide deposits. Analyses of lake bottom and influent waters suggested that frequent resampling of the sites would be required throughout the year to permit meaningful interpretation of the effect of water composition of concretions. Analyses of sediment cores from 20 concretion sites indicated an upward increase in Fe and Mn and in the Mn/Fe ratio, consistent with the model of upward migration of the elements, where Mn is more mobile than Fe. This study concludes that a considerable proportion of the elements have been supplied to the Shebandowan concretions via the diagenetic process; generally a minor fraction of the elements has been abstracted directly from the superjacent water.

Uptake of soluble phosphate by activated sludge /

"Supported by Federal Water Pollution Control Administration ... research fellowship F1-WP-21, 616, and Ford Foundation."

Seasonal patterns of growth, dehydrins and water-soluble carbohydrates in genotypes of Dactylis glomerata varying in summer dormancy

Background and Aims Summer dormancy in perennial grasses has been studied inadequately, despite its potential to enhance plant survival and persistence in Mediterranean areas. The aim of the present work was to characterize summer dormancy and dehydration tolerance in two cultivars of Dactylis glomerata (dormant 'Kasbah', non-dormant 'Oasis') and their hybrid using physiological indicators associated with these traits. Methods Dehydration tolerance was assessed in a glasshouse experiment, while seasonal metabolic changes which produce putative protectants for drought, such as carbohydrates and dehydrins that might be associated with summer dormancy, were analysed in the field. Key Results The genotypes differed in their ability to survive increasing soil water deficit: lethal soil water potential (ψ(s)) was -3(.)4 MPa for 'Kasbah' (although non-dormant), -1(.)3 MPa for 'Oasis', and -1(.)6 MPa for their hybrid. In contrast, lethal water content of apices was similar for all genotypes (approx. 0(.)45 g H2O g d. wt(-1)), and hence the greater survival of 'Kasbah' can be ascribed to better drought avoidance rather than dehydration tolerance. In autumn-sown plants, 'Kasbah' had greatest dormancy, the hybrid was intermediate and 'Oasis' had none. The more dormant the genotype, the lower the metabolic activity during summer, and the earlier the activity declined in spring. Decreased monosaccharide content was an early indicator of dormancy induction. Accumulation of dehydrins did not correlate with stress tolerance, but dehydrin content was a function of the water status of the tissues, irrespective of the soil moisture. A protein of approx. 55 kDa occurred in leaf bases of the most dormant cultivar even in winter. Conclusions Drought avoidance and summer dormancy are correlated but can be independently expressed. These traits are heritable, allowing selection in breeding programmes.

The effect of selected water-soluble excipients on the dissolution of paracetamol and ibuprofen

The purpose of this investigation was to study the dissolution behavior of paracetamol and ibuprofen in the presence of a range of selected potential excipients. First, a pH-solubility profile was generated for both drugs, and the effect of changing hydrodynamic conditions on the intrinsic dissolution rate was investigated. It was established that both drugs dissolved according to the diffusion-layer model. Paracetamol solubility (approximately 20.3 mg mL -1) did not vary from pH 1.2-8.0, corresponding to the in vivo range in the gastrointestinal tract. Ibuprofen had an intrinsic solubility of approximately 0.06 mg mL-1, and pKa was calculated as 4.4. Second, the effects of selected potential excipients (lactose, potassium bicarbonate, sodium bicarbonate, sodium chloride, and tartaric acid) were evaluated by measuring the effect of the inclusion of each additive in the dissolution medium on drug solubility, drug intrinsic dissolution rate, and solution viscosity. The results were evaluated using the diffusion-layer model, and it was determined that for paracetamol, the collected data fitted the model for all the excipients studied. For ibuprofen, it was found that there were differences between the excipients that raised the solution pH above the pK a to those that did not. For the excipients raising the pH above the pKa, the effect on intrinsic dissolution rate was not as high as that expected from the change in drug solubility. It was postulated that this might be due to lack of penetration of the excipient into the drug boundary layer microenvironment. Formulators may calculate the effect of adding an excipient based on solubility increases but may not find the dissolution rate improvement expected. Copyright © 2005 Taylor & Francis Inc.

Plasma vascular endothelial growth factor, angiopoietin-2, and soluble angiopoietin receptor tie-2 in diabetic retinopathy:effects of laser photocoagulation and angiotensin receptor blockade

Background: Proliferative diabetic retinopathy (PDR) may be a response to abnormal angiogenic growth factors such as vascular endothelial growth factor (VEGF), angiopoietin-2 (Ang-2), and the soluble angiopoietin receptor tie-2. The authors hypothesised the following: (a) there are differences in plasma levels of these growth factors in different grades of diabetic retinopathy; and (b) that the effects of intervention with panretinal laser photocoagulation (PRP) for PDR, and angiotensin receptor blockade (using eprosartan) for patients with other grades of diabetic retinopathy will be to reduce levels of the growth factors. Methods: Cross sectional and interventional study (using PRP and eprosartan) in diabetic patients. VEGF, Ang-2, and tie-2 were measured by ELISA. Results: VEGF (p<0.001) and Ang-2 levels (p<0.001) were significantly higher in 93 diabetic patients compared to 20 healthy controls, with the highest levels in grade 2 and grade 3 diabetic retinopathy (p<0.05). Tie-2 was lower in diabetics compared to controls (p = 0.008), with no significant differences between the diabetic subgroups. Overall, VEGF significantly correlated with Ang-2 (p<0.001) and tie-2 (p = 0.004) but the correlation between Ang-2 and tie-2 levels was not significant (p = 0.065). Among diabetic patients only, VEGF levels were significantly correlated with Ang-2 (p<0.001) and tie-2 (p<0.001); the correlation between Ang-2 and tie-2 levels was also significant (p<0.001). There were no statistically significant effects of laser photocoagulation on plasma VEGF, Ang-2, and tie-2 in the 19 patients with PDR, or any effects of eprosartan in the 28 patients with non-proliferative diabetic retinopathy. Conclusion: Increased plasma levels of VEGF and Ang-2, as well as lower soluble tie-2, were found in diabetic patients. The highest VEGF and Ang-2 levels were seen among patients with pre-proliferative and proliferative retinopathy, but there was no relation of tie-2 to the severity of retinopathy. As the majority of previous research into Ang-2 and tie-2 has been in relation to angiogenesis and malignancy, the present study would suggest that Ang-2 and tie-2 may be used as potential indices of angiogenesis in diabetes mellitus (in addition to VEGF) and may help elucidate the role of the angiopoietin/tie-2 system in this condition.

Elevated placental soluble vascular endothelial growth factor receptor-1 inhibits angiogenesis in preeclampsia

Preeclampsia is an inflammatory disorder in which serum levels of vascular endothelial growth factor (VEGF) and its soluble receptor-1 (sVEGFR-1, also known as sFlt-1) are elevated. We hypothesize that VEGF and placenta growth factor (PlGF) are dysregulated in preeclampsia due to high levels of sVEGFR-1, which leads to impaired placental angiogenesis. Analysis of supernatants taken from preeclamptic placental villous explants showed a four-fold increase in sVEGFR-1 than normal pregnancies, suggesting that villous explants in vitro retain a hypoxia memory reflecting long-term fetal programming. The relative ratios of VEGF to sVEGFR-1and PlGF to sVEGFR-1 released from explants decreased by 53% and 70%, respectively, in preeclampsia compared with normal pregnancies. Exposure of normal villous explants to hypoxia increased sVEGFR-1 release compared with tissue normoxia (P<0.001), as did stimulation with tumor necrosis factor-α (P<0.01). Conditioned medium (CM) from normal villous explants induced endothelial cell migration and in vitro tube formation, which were both attenuated by pre-incubation with exogenous sVEGFR-1 (P<0.001). In contrast, endothelial cells treated with preeclamptic CM showed substantially reduced angiogenesis compared withnormal CM (P<0.001), which was not further decreased by the addition of exogenous sVEGFR-1, indicating a saturation of the soluble receptor.Removal of sVEGFR-1 by immunoprecipitation from preeclamptic CM significantly restored migration (P<0.001) and tube formation (P<0.001) to levels comparable to that induced by normal CM, demonstrating that elevated levels of sVEGFR-1 in preeclampsia are responsible for inhibiting angiogenesis. Our finding demonstrates the dysregulation of the VEGF/PlGF axis in preeclampsiaand offers an entirely new therapeutic approach to its treatment.

Heparin elevates circulating soluble fms-like tyrosine kinase-1 immunoreactivity in pregnant women receiving anticoagulation therapy

Background—Alterations in circulating levels of pro- and antiangiogenic factors have been associated with adverse pregnancy outcomes. Heparin is routinely administered to pregnant women, but without clear knowledge of its impact on these factors. Methods and Results—We conducted a longitudinal study of 42 pregnant women. Twenty-one women received prophylactic heparin anticoagulation, and 21 healthy pregnant women served as controls. Compared with gestational age-matched controls, heparin treatment was associated with increased circulating levels of soluble fms-like tyrosine kinase-1 (sFlt-1) in the third trimester (P<0.05), in the absence of preeclampsia, placental abruption, or fetal growth restriction. Heparin had no effect on circulating levels of vascular endothelial growth factor, placenta growth factor, or soluble endoglin as assessed by ELISA. In vitro, low-molecular weight and unfractionated heparins stimulated sFlt-1 release from placental villous explants, in a dose- and time-dependent manner. This effect was not due to placental apoptosis, necrosis, alteration in protein secretion, or increased transcription. Western blot analysis demonstrated that heparin induced shedding of the N-terminus of Flt-1 both in vivo and in vitro as indicated by a predominant band of 100–112 kDa. By using an in vitro angiogenesis assay, we demonstrated that serum of heparin-treated cases inhibited both basal and vascular endothelial growth factor-induced capillary-like tube formation. Conclusions—Heparin likely increases the maternal sFlt-1 through shedding of the extracellular domain of Flt-1 receptor. Our results imply that upregulation of circulating sFlt-1 immunoreactivity in pregnancy is not always associated with adverse outcomes, and that heparin's protective effects, if any, cannot be explained by promotion of angiogenesis.

Upregulation of urotensin II receptor in preeclampsia causes in vitro placental release of soluble vascular endothelial growth factor receptor 1 in hypoxia

Preeclampsia is a hypertensive disorder of pregnancy caused by abnormal placental function, partly because of chronic hypoxia at the utero-placental junction. The increase in levels of soluble vascular endothelial growth factor receptor 1, an antiangiogenic agent known to inhibit placental vascularization, is an important cellular factor implicated in the onset of preeclampsia. We investigated the ligand urotensin II (U-II), a potent endogenous vasoconstrictor and proangiogenic agent, for which levels have been reported to increase in patients with preeclampsia. We hypothesized that an increased sensitivity to U-II in preeclampsia might be achieved by upregulation of placental U-II receptors. We further investigated the role of U-II receptor stimulation on soluble vascular endothelial growth factor receptor 1 release in placental explants from diseased and normal patients. Immunohistochemistry, real-time PCR, and Western blotting analysis revealed that U-II receptor expression was significantly upregulated in preeclampsia placentas compared with controls (P<0.01). Cellular models of syncytiotrophoblast and vascular endothelial cells subjected to hypoxic conditions revealed an increase in U-II receptor levels in the syncytiotrophoblast model. This induction is regulated by the transcriptional activator hypoxia-inducible factor 1a. U-II treatment is associated with increased secretion of soluble vascular endothelial growth factor receptor 1 only in preeclamptic placental explants under hypoxia but not in control conditions. Interestingly, normal placental explants did not respond to U-II stimulation.

Serum procalcitonin, interleukin-6, soluble intercellular adhesin molecule-1 and IgG to short-chain exocellular lipoteichoic acid as predictors of infection in total joint prosthesis revision

The diagnosis of prosthetic joint infection and its differentiation from aseptic loosening remains problematic. The definitive laboratory diagnostic test is the recovery of identical infectious agents from multiple intraoperative tissue samples; however, interpretation of positive cultures is often complex as infection is frequently associated with low numbers of commensal microorganisms, in particular the coagulase-negative staphylococci (CNS). In this investigation, the value of serum procalcitonin (PCT), interleukin-6 (IL-6) and soluble intercellular adhesion molecule-1 (sICAM-1) as predictors of infection in revision hip replacement surgery is assessed. Furthermore, the diagnostic value of serum IgG to short-chain exocellular lipoteichoic acid (sce-LTA) is assessed in patients with infection due to CNS. Presurgical levels of conventional serum markers of infection including C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and white blood cell count (WBC) is also established. Forty-six patients undergoing revision hip surgery were recruited with a presumptive clinical diagnosis of either septic (16 patients) or aseptic loosening (30 patients). The diagnosis was confirmed microbiologically and levels of serum markers were determined. Serum levels of IL-6 and sICAM-1 were significantly raised in patients with septic loosening (P=0.001 and P=0.0002, respectively). Serum IgG to sce-LTA was elevated in three out of four patients with infection due to CNS. In contrast, PCT was not found to be of value in differentiating septic and aseptic loosening. Furthermore, CRP, ESR and WBC were significantly higher (P=0.0001, P=0.0001 and P=0.003, respectively) in patients with septic loosening. Serum levels of IL-6, sICAM-1 and IgG to sce-LTA may provide additional information to facilitate the diagnosis of prosthetic joint infection.

(Table 3) Sulfur and oxygen isotopic composition of cold water soluble sulfate, and strontium content and isotopic composition of upper Miocene evaporite samples recovered from DSDP Site 23-227

The origin of three Red Sea submarine brine pools was investigated by analysis of the S and O isotope ratios of dissolved sulfate and Sr isotope ratios of dissolved Sr in the brines. Sulfur and O isotope ratios of sulfate and Sr isotope ratios of evaporitic source rocks for the brines were measured for comparison. The S, O and Sr isotope ratios of evaporites recovered from DSDP site 227 are consistent with an upper Miocene evaporites age. The Valdivia Deep brine formed by karstic dissolution of Miocene evaporites by overlying seawater and shows no signs of hydrothermal input. The Suakin Deep brines are derived from, or have isotopically exchanged with Miocene or older evaporites. There has been only minor dilution of the brine by overlying seawater. Strontium isotope ratios of Suakin brine may indicate addition of a minor (15%) amount of volcanic Sr to the brine, but there is no evidence of high temperature brine-rock interaction. The sulfate in the Atlantis II brine was apparently derived from seawater. The O isotope ratio of sulfate in the present Atlantis II brine could reflect isotopic exchange between seawater sulfate and the brine at approximately 255°C. Approximately 30% of the Sr in the Atlantis II brine is derived from the underlying basalt, probably by hydrothermal leaching. Atlantis II brine is the only known example from the Red Sea which has a significant high-temperature hydrothermal history.

(Table 5, page 626), Tin and main metal elements composition of ferromanganese concretions (acid-soluble fractions)

Concentrations of tin in sea water decreased from estuarine and shelf (0.02-0.04 µg/kg) to surface Atlantic waters (0.009 µg/kg). Mean contents (ppm) in other materials included: ultramafic rocks, 0.8; basalts, 1.7; silicic rocks, 2.5; red clays, 3.4; amphibolites, 1.2. Oceanic ferromanganese deposits contained from 0.2 to 5.8 ppm; tin and cobalt contents were correlated.

Immunoregulatory soluble CTLA-4 modifies effector T cell responses in systemic lupus erythematosus

Acknowledgments This work was supported by Arthritis Research UK (Grant no. 19282). We are grateful to Dr. Nick Fluck for his invaluable support in recruiting patients for the study, and Mrs. Vivien Vaughan for her invaluable expertise in recruiting study participants and maintaining ethical documentation.