Recognition and clearance of methoxypoly(ethyleneglycol)2000-grafted liposomes by macrophages with enhanced phagocytic capacity. Implications in experimental and clinical oncology.

Intravenous injection of an endotoxin-free solution of poloxamine-908 to rats can enhance the phagocytic clearance capacity of tissue macrophages, particularly those of the liver and the spleen. Such stimulated cells were able to clear a significant portion of intravenously injected methoxypoly(ethyleneglycol)2000 liposomes (mean size of 87 nm), labelled with technetium-99m via the N-hydroxysuccinimidyl hydrazine nicotinate hydrochloride derivative of distearoyl phosphatidylethanolamine, within 4 h post administration. These liposomes, otherwise, exhibit long circulatory behaviour in control animals, with poor localization to the liver and spleen. We suggest that such technetium-99m-labelled engineered vesicles may be of aid for detection of the liver and spleen macrophages with enhanced phagocytic clearance capacity by gamma scintigraphy. Alterations in the phagocytic activity of liver and spleen macrophages is known to occur during cancer. Therefore, such diagnostic procedures may prove useful for patient selection or for monitoring the progress of treatment with long circulating nanoparticles carrying anti-cancer agents, thus minimizing damage to this important line of body's defence cells, and are discussed.     

Anaerobic degradation of papermill sludge in a two-phase digester containing rumen microorganisms and colonized polyurethane foam

Summary The use of reticulated polyurethane foam as a support material for the immobilization of methanogenic associations and its application to the anaerobic treatment of fine particulate solid wastes was investigated. The colonization of polyurethane support particles in a continuous upflow reactor fed on a mixture of acetate, propionate and butyrate, was both rapid and dense. The combination of rumen microorganisms and colonized support particles in a two-phase digester resulted in an efficient anaerobic decomposition of papermill sludge.     

Two technetium-99m-labeled cholecystokinin-8 (CCK8) peptides for scintigraphic imaging of CCK receptors

A broad spectrum of radiolabeled peptides with high affinity for receptors expressed on tumor cells is currently under preclinical and clinical investigation for scintigraphic imaging and radionuclide therapy. The present paper evaluates two (99m)Tc-labeled forms of the C-terminal octapeptide of cholecystokinin (CCK8): sulfated (s)CCK8, with high affinity for CCK1 and CCK2 receptors, and nonsulfated (ns)CCK8, with high affinity for CCK2 receptors but low affinity for CCK1 receptors. Peptides were conjugated with the bifunctional chelator N-hydroxysuccinimidyl hydrazino niconitate (s-HYNIC). (99m)Tc-labeling, performed in the presence of nicotinic acid and tricine, was highly efficient (approximately 95%) and yielded products with a high specific activity (approximately 700 Ci/mmol) and good stability (approximately 5% release of radiolabel during 16 h incubation in phosphate buffered saline at 37 degrees C). Chinese hamster ovary cells stably expressing the CCK1 receptor (CHO-CCK1 cells) internalized approximately 3% of added (99m)Tc-sCCK8 per confluent well during 2 h at 37 degrees C. Internalization was effectively blocked by excess unlabeled sCCK8. CHO-CCK1 cells did not internalize (99m)Tc-nsCCK8. Displacement of (99m)Tc-sCCK8 and -nsCCK8 by unlabeled CCK-8 (performed at 0 degrees C to prevent internalization) revealed 50% inhibitory concentrations (IC(50)) of 8 nM and >1 microM, respectively. CHO-CCK2 cells internalized approximately 25% and approximately 5% of added (99m)Tc-sCCK8 and -nsCCK8, respectively. In both cases internalization was blocked by excess unlabeled peptide. IC(50) values for the displacement of (99m)Tc-sCCK8 and -nsCCK8 were 3 nM and 10 nM, respectively. CHO-CCK1 cell-derived tumors present in one flank of athymic mice accumulated 2.0% of injected (99m)Tc-sCCK8 per gram tissue at 1 h postinjection. This value decreased to 0.6% following coinjection with excess unlabeled peptide. Uptake of (99m)Tc-nsCCK8 was low (0.2%) and not did change by excess unlabeled peptide (0.3%). Accumulation of (99m)Tc-sCCK8 and -nsCCK8 by CHO-CCK2 cell-derived tumors (present in the other flank) amounted to 4.2% and 0.6%, respectively. In both cases uptake was significantly reduced by excess unlabeled peptide to 1.0% and 0.4% for sCCK8 and nsCCK8, respectively. Accumulation of (99m)Tc-sCCK8 was also high in pancreas (11.7%), stomach (2.0%), and kidney (2.1%), whereas uptake of (99m)Tc-nsCCK8 was high in stomach (0.7%) and kidney (1.4%). Both radiolabeled peptides showed a rapid blood clearance. In conclusion, these data show that CCK8 analogues can be efficiently labeled with (99m)Tc using s-HYNIC as chelator and nicotinic acid/tricine as coligand system without compromising receptor binding. Furthermore, the present study demonstrates that CCK1 tumors hardly accumulate (99m)Tc-nsCCK8, CCK2 tumors accumulate 2 times more (99m)Tc-sCCK8 than CCK1 tumors, and CCK2 tumors accumulate 15 times more (99m)Tc-sCCK8 than (99m)Tc-nsCCK8. Although accumulation in some nontarget organs was also higher with (99m)Tc-sCCK8, this may not reflect the human situation due to a different receptor expression pattern in humans as compared to mice. Therefore, further studies are warranted to investigate the possible use of (99m)Tc-sCCK8 for scintigraphic imaging of CCK receptor-positive tumors in humans.     

Radiolabeled liposomes for scintigraphic imaging

Liposomes have been investigated extensively as carriers for drugs in attempts to achieve selective deposition and/or reduced toxicity. Liposomes radiolabeled with gamma emitters such as (67)Ga, (111)In and (99m)Tc, can be used for imaging purposes. Liposomes as formulated in the past, are rapidly taken up by cells of the mononuclear phagocyte system (MPS), primarily those located in liver and spleen. The recent development of long-circulating liposomes (LCLs), yielded liposomes that oppose recognition by the MPS. The development of these LCLs with enhanced circulatory half-lives has broadened the potential of liposomes to scintigraphically visualize pathologic processes in vivo. Liposomes have been proposed for tumor imaging, infection imaging and blood pool imaging. Strategies have been developed that allow rapid, easy and efficient labeling of preformed liposomes with (111)In and (99m)Tc. There is now a vast body of preclinical evidence showing that LCLs can be used to image a wide variety of tumors as well as inflammatory lesions. The first studies in patients show that radiolabeled liposomes can image tumor and inflammatory lesions with good sensitivity and good specificity. Here, the present status of liposome-based radiopharmaceuticals for scintigraphic application is reviewed.     

Preparation and evaluation of glycosylated arginine-glycine-aspartate (RGD) derivatives for integrin targeting

Arginine-glycine-aspartate (RGD) derivatives were prepared by a combination of solid-phase and solution-phase synthesis for selective targeting of alpha vbeta 3 integrin expressed in tumors. In order to evaluate the value of a triazole moiety as a proposed amide isostere, the side chain glycosylated cyclic RGD ( cRGD) peptides were synthesized with either a natural amide linkage or a triazole. Affinity of the cRGD constructs for the alpha vbeta 3 integrin was determined in a solid-phase competitive binding assay, showing strong similarity in binding affinity for each of the compounds under evaluation. Furthermore, the in vivo tumor targeting potential of glycosylated cRGD peptides, linked via amide or triazole, was investigated by determining the biodistribution of (125)I-labeled derivatives in mice with tumors expressing alpha vbeta 3. All of the cyclic RGD derivatives showed preferential uptake in the subcutaneous tumors, with the highest tumor-to-blood ratio measured for the triazole-linked glycosylated derivative. The results of the present study are a clear indication of the value of the triazole moiety as a suitable amide isostere in the development of glycosylated peptides as pharmaceuticals.     

Airway cellularity, lipid laden macrophages and microbiology of gastric juice and airways in children with reflux oesophagitis

Background and methods

Human metapneumovirus (hMPV) is a recently discovered respiratory virus associated with bronchiolitis, pneumonia, croup and exacerbations of asthma. Since respiratory viruses are frequently detected in patients with acute exacerbations of COPD (AE-COPD) it was our aim to investigate the frequency of hMPV detection in a prospective cohort of hospitalized patients with AE-COPD compared to patients with stable COPD and to smokers without by means of quantitative real-time RT-PCR.

Results

We analysed nasal lavage and induced sputum of 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. HMPV was detected in 3/130 (2.3%) AE-COPD patients with a mean of 6.5 × 10⁵ viral copies/ml in nasal lavage and 1.88 × 10⁵ viral copies/ml in induced sputum. It was not found in patients with stable COPD or smokers without COPD.

Conclusion

HMPV is only found in a very small number of patients with AE-COPD. However it should be considered as a further possible viral trigger of AE-COPD because asymptomatic carriage is unlikely.     

Bioavailability of organic matter in a freshwater estuarine sediment: long<Emphasis Type="Bold">-</Emphasis>term degradation experiments with and without nitrate supply

Organic carbon degradation experiments were carried out using flow-through reactors with sediments collected from an intertidal freshwater marsh of an eutrophic estuary (The Scheldt, Belgium). Concentrations of nitrate, nitrite, dissolved inorganic carbon (DIC), dissolved organic carbon, methane, dissolved cations (Ca²⁺, Mg²⁺, Na⁺ and K⁺), total dissolved Fe, phosphate and alkalinity were measured in the outflow solutions from reactors that were supplied with or without the terminal electron acceptor nitrate. Organic carbon mineralization rates were computed from the release rates of DIC after correcting for the contribution of carbonate mineral dissolution. The experiments ran for several months until nitrate reducing activity could no longer be detected. In the reactors supplied with nitrate, 10–13% of the bulk sedimentary organic carbon (SOC) was mineralized by the end of the experiments. In reactors receiving no nitrate, only 3–9% of the initial SOC was mineralized. Organic matter utilization by nitrate reducers could be described as the simultaneous degradation of two carbon pools with different maximum oxidation rates and half-saturation constants. Even when nitrate was supplied in non-limiting concentrations about half of the carbon mineralization in the reactors was due to fermentative processes, rather than being coupled to nitrate respiration. Fermentation may thus be responsible for a large fraction of the DIC efflux from organic-rich, nearshore sediments.     

Bacterial Dissimilatory Reduction of Arsenic(V) to Arsenic(III) in Anoxic Sediments

Incubation of anoxic salt marsh sediment slurries with 10 mM As(V) resulted in the disappearance over time of the As(V) in conjunction with its recovery as As(III). No As(V) reduction to As(III) occurred in heat-sterilized or formalin-killed controls or in live sediments incubated in air. The rate of As(V) reduction in slurries was enhanced by addition of the electron donor lactate, H(inf2), or glucose, whereas the respiratory inhibitor/uncoupler dinitrophenol, rotenone, or 2-heptyl-4-hydroxyquinoline N-oxide blocked As(V) reduction. As(V) reduction was also inhibited by tungstate but not by molybdate, sulfate, or phosphate. Nitrate inhibited As(V) reduction by its action as a preferred respiratory electron acceptor rather than as a structural analog of As(V). Nitrate-respiring sediments could reduce As(V) to As(III) once all the nitrate was removed. Chloramphenicol blocked the reduction of As(V) to As(III) in nitrate-respiring sediments, suggesting that nitrate and arsenate were reduced by separate enzyme systems. Oxidation of [2-(sup14)C]acetate to (sup14)CO(inf2) by salt marsh and freshwater sediments was coupled to As(V). Collectively, these results show that reduction of As(V) in sediments proceeds by a dissimilatory process. Bacterial sulfate reduction was completely inhibited by As(V) as well as by As(III).     

Coexistence of aerobic chemotrophic and anaerobic phototrophic sulfur bacteria under oxygen limitation

Abstract: The aerobic chemotrophic sulfur bacterium Thiobacillus thioparus T5 and the anaerobic phototrophic sulfur bacterium Thiocapsa roseopersicina M1 were co-cultured in continuously illuminated chemostats at a dilution rate of 0.05 h⁻¹. Sulfide was the only externally supplied electron donor, and oxygen and carbon dioxide served as electron acceptor and carbon source, respectively. Steady states were obtained with oxygen supplies ranging from non-limiting amounts (1.6 mol O₂ per mol sulfide, resulting in sulfide limitation) to severe limitation (0.65 mol O₂ per mol sulfide). Under sulfide limitation Thiocapsa was competitively excluded by Thiobacillus and washed out. Oxygen/sulfide ratios between 0.65 and 1.6 resulted in stable coexistence. It could be deduced that virtually all sulfide was oxidized by Thiobacillus . The present experiments showed that Thiocapsa is able to grow phototrophically on the partially oxidized products of Thiobacillus . In pure Thiobacillus cultures in steady state extracellular zerovalent sulfur accumulated, in contrast to mixed cultures. This suggests that a soluble form of sulfur at the oxidation state of elemental sulfur is formed by Thiobacillus as intermediate. As a result, under oxygen limitation colorless sulfur bacteria and purple sulfur bacteria do not competitively exclude each other but can coexist. It was shown that its ability to use partially oxidized sulfur compounds, formed under oxygen limiting conditions by Thiobacillus , helps explain the bloom formation of Thiocapsa in marine microbial mats.