Agents Dextran Research Articles

Magnetic resonance imaging detection of an uncommon granuloma formation after endothelial progenitor cells transplantation

Magnetic resonance imaging (MRI) has been recognized as a non-invasive technique for visualizing the ultrastructure of a tissue at high resolution. The work reported here showed the utility of MRI for visualizing the fate of EPCs, and demonstrate how it can be used to further our understanding of angiogenesis mechanisms.The recently developed contrast agent dextran mono-N-succinimidyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate-gadolinium³⁺ (Dex-DOTA-Gd³⁺) was used to label endothelial progenitor cells (EPCs). The faith of the transplanted labeled cells in the rat models of ischemic hind limbs was studied by images obtained by MRI.The pattern of migration of Dex-DOTA-Gd³⁺-labeled EPCs could be observed and tracked by MRI for a long time and analyzed the change in the migration of the labeled cells. The ability of Dex-DOTA-Gd³⁺ to provide a clear pattern of cell migration to the limb was confirmed and most importantly, a different behavioral pattern was identified in the migration of labeled cells when an anomaly appeared in the MRI acquisition images 5 days post transplantation.

Loss of TMF/ARA160 Protein Renders Colonic Mucus Refractory to Bacterial Colonization and Diminishes Intestinal Susceptibility to Acute Colitis

TMF/ARA160 is a Golgi-associated protein with several cellular functions, among them direction of the NF-κB subunit, p65 RelA, to ubiquitination and proteasomal degradation in stressed cells. We sought to investigate the role of TMF/ARA160 under imposed stress conditions in vivo. TMF(-/-) and wild-type (WT) mice were treated with the ulcerative agent dextran sulfate sodium (DSS), and the severity of the inflicted acute colitis was determined. TMF(-/-) mice were found to be significantly less susceptible to DSS-induced colitis, with profoundly less bacterial penetration into the colonic epithelia. Surprisingly, unlike in WT mice, no bacterial colonies were visualized in colons of healthy untreated TMF(-/-) mice, indicating the constitutive resistance of TMF(-/-) colonic mucus to bacterial retention and penetration. Gene expression analysis of colon tissues from unchallenged TMF(-/-) mice revealed 5-fold elevated transcription of the muc2 gene, which encodes the major component of the colonic mucus gel, the MUC2 mucin. Accordingly, the morphology of the colonic mucus in TMF(-/-) mice was found to differ from the mucus structure in WT colons. The NF-κB subunit, p65, a well known transcription inducer of muc2, was up-regulated significantly in TMF(-/-) intestinal epithelial cells. However, this did not cause spontaneous inflammation or increased colonic crypt cell proliferation. Collectively, our findings demonstrate that absence of TMF/ARA160 renders the colonic mucus refractory to bacterial colonization and the large intestine less susceptible to the onset of colitis.

Protein Disorder Prevails under Crowded Conditions

Crowding caused by the high concentrations of macromolecules in the living cell changes chemical equilibria, thus promoting aggregation and folding reactions of proteins. The possible magnitude of this effect is particularly important with respect to the physiological structure of intrinsically disordered proteins (IDPs), which are devoid of well-defined three-dimensional structures in vitro. To probe this effect, we have studied the structural state of three IDPs, α-casein, MAP2c, and p21(Cip1), in the presence of the crowding agents Dextran and Ficoll 70 at concentrations up to 40%, and also the small-molecule osmolyte, trimethylamine N-oxide (TMAO), at concentrations up to 3.6 M. The structures of IDPs under highly diluted and crowded conditions were compared by a variety of techniques, fluorescence spectroscopy, acrylamide quenching, 1-anilino-8-naphthalenesulfonic acid (ANS) binding, fluorescence correlation spectroscopy (FCS), and far-UV and near-UV circular dichroism (CD) spectroscopy, which allow us to visualize various levels of structural organization within these proteins. We observed that crowding causes limited structural changes, which seem to reflect the functional requirements of these IDPs. α-Casein, a protein of nutrient function in milk, changes least under crowded conditions. On the other hand, MAP2c and, to a lesser degree, p21(Cip1), which carry out their functions by partner binding and accompanying partially induced folding, show signs of local structuring and also some global compaction upon crowded conditions, in particular in the presence of TMAO. The observations are compatible with the possible preformation of binding-competent conformations in these proteins. The magnitude of these changes, however, is far from that of the cooperative folding transitions elicited by crowding in denatured globular proteins; i.e., these IDPs do remain in a state of rapidly interconverting structural ensemble. Altogether, our results underline that structural disorder is the physiological state of these proteins.

Quantification of Titin Based Viscosity: Temperature, Lattice Compression and Integrative Physiology

Viscosity is proposed to modulate diastolic function, but there is only limited understanding of the source(s) of viscosity. In-vitro experiments have shown that the proline-glutamic acid-valine-lysine (PEVK) rich element of titin binds actin, causing a viscous force in the sarcomere. It is unknown whether this mechanism contributes to viscosity in-vivo. Therefore, we sought to test, via an integrative physiological study on a unique PEVK-knockout (KO) mouse, the hypothesis that PEVK-actin interaction causes significant cardiac viscosity and is important in-vivo.METHODS AND RESULTS: Both skinned cardiomyocytes and papillary muscle fibers were isolated from wildtype (WT) and PEVK KO mice and viscosity was examined using stretch-hold-release and sinusoidal analysis. We previously found that viscosity is reduced by ∼60% in KO myocytes and ∼50% muscle fibers at room temperature (24oC). Inhibition by blebbistatin reveals that actomyosin interaction is not present at room temperature but contributes to viscosity at physiologic temperature (37oC). We also examined the passive contribution of lattice compression and temperature while inhibiting actomyosin interactions with blebbistatin. Lattice compression using the osmotic agent Dextran T500 enhances viscosity in WT but not KO tissues but increasing temperature alone does not significantly increase viscosity. We also studied intact isolated hearts via a Langendorff perfused volume-controlled system. Stretch-hold protocols and sinusoidal frequency protocols indicated that KO hearts have a ∼30% reduction in viscosity. Finally, transmitral Doppler echocardiography and kinematic modeling was utilized to examine left ventricular viscosity in-vivo. Quantifying viscosity with both traditional and kinematic echocardiographic measurements suggest a ∼40% decrease in viscosity in the KO left ventricle.CONCLUSIONS: This integrative study is the first to quantify the consequences of a specific molecular (PEVK-actin) viscosity in-vivo and physiologic modulation of this passive viscosity by lattice spacing.

The AOM/DSS murine model for the study of colon carcinogenesis: From pathways to diagnosis and therapy studies

Colorectal cancer (CRC) is a major health problem in industrialized countries. Although inflammation-linked carcinogenesis is a well accepted concept and is often observed within the gastrointestinal tract, the underlying mechanisms remain to be elucidated. Inflammation can indeed provide initiating and promoting stimuli and mediators, generating a tumour-prone microenvironment. Many murine models of sporadic and inflammation-related colon carcinogenesis have been developed in the last decade, including chemically induced CRC models, genetically engineered mouse models, and xenoplants. Among the chemically induced CRC models, the combination of a single hit of azoxymethane (AOM) with 1 week exposure to the inflammatory agent dextran sodium sulphate (DSS) in rodents has proven to dramatically shorten the latency time for induction of CRC and to rapidly recapitulate the aberrant crypt foci–adenoma–carcinoma sequence that occurs in human CRC. Because of its high reproducibility and potency, as well as the simple and affordable mode of application, the AOM/DSS has become an outstanding model for studying colon carcinogenesis and a powerful platform for chemopreventive intervention studies. In this article we highlight the histopathological and molecular features and describe the principal genetic and epigenetic alterations and inflammatory pathways involved in carcinogenesis in AOM/DSS–treated mice; we also present a general overview of recent experimental applications and preclinical testing of novel therapeutics in the AOM/DSS model.

Distinct fucosylation of M cells and epithelial cells by Fut1 and Fut2, respectively, in response to intestinal environmental stress

The intestinal epithelium contains columnar epithelial cells (ECs) and M cells, and fucosylation of the apical surface of ECs and M cells is involved in distinguishing the two populations and in their response to commensal flora and environmental stress. Here, we show that fucosylated ECs (F-ECs) were induced in the mouse small intestine by the pro-inflammatory agents dextran sodium sulfate and indomethacin, in addition to an enteropathogen derived cholera toxin. Although F-ECs showed specificity for the M cell-markers, lectin Ulex europaeus agglutinin-1 and our monoclonal antibody NKM 16-2-4, these cells also retained EC-phenotypes including an affinity for the EC-marker lectin wheat germ agglutinin. Interestingly, fucosylation of Peyer’s patch M cells and F-ECs was distinctly regulated by α(1,2)fucosyltransferase Fut1 and Fut2, respectively. These results indicate that Fut2-mediated F-ECs share M cell-related fucosylated molecules but maintain distinctive EC characteristics, Fut1 is, therefore, a reliable marker for M cells.

Abstract ED06-04: The microbiota and development of colitis-associated colorectal cancer

Abstract The human intestinal microbiota is a community of 1013-1014 microorganisms that reside in the intestine and normally participate in a symbiotic relationship with their eukaryotic host. The microbiome contains a wealth of information, encompassing 150 fold as many genes as the human genome. Recent studies have hinted that a core human microbiome exists at the gene level, with a large number of microbial genes and pathways shared among individuals. Deviations from this core microbiome could potentially affect human health and promote disease state. Thus characterizing the vast intestinal microbial community present in healthy as well as pathologic conditions will likely revolutionize our conception of bacteria/host interactions and identify new physiological processes influenced by the microbiota. Already the microbiota has been linked to cardiac development, angiogenesis, innate and adaptive immunity, metabolism, nutrient acquisition, and gastrointestinal development and homeostasis. Furthermore, alterations in the microbial community are associated with multiple diseases, including obesity, fatty liver disease, type 1 and type 2 diabetes, kidney disease, arthritis and inflammatory bowel diseases (IBD). Eukaryotes are equipped with an elegant repertoire of innate receptors each recognizing specific conserved microbial patterns present on various microorganisms, such as components of bacterial cell walls, locomotion system or nucleic acids. These microbial sensors are termed pattern recognition receptors (PRR) and include retinoic acid inducible gene-I like RNA helicases (RLH), C-type lectin receptors (CLR), Nucleotide-binding domain leucine-rich repeat proteins (NLR; also known as Nod-like receptors), and Toll-like receptors (TLR). Although the etiology of colitis-associated colorectal cancer remains elusive, innate host sensors have been shown to modulate the pathogenesis. Indeed, using mice exposed to the colon-specific carcinogen azoxymethane (AOM) compound and to the mucosal-damaging agent dextran sulfate sodium (DSS), numerous studies have revealed the differential contribution of innate sensors to CAC. For example, the NLR protein NOD1, a close relative of the IBD susceptible gene NOD2 protects against the development of colitis and colorectal cancer. Similarly, the adaptor protein MyD88 frequently utilized by TLR family members is protective in the context of AOM/DSS-induced colorectal cancer. Furthermore, the intracellular sensor NLRP3 also appears protective against colitis and CAC. In contrast, TLR4 signaling promotes the development of colorectal cancer in the AOM/DSS model. Although informative, these studies only capture the impact of various innate host responses on the development of colorectal cancer. The true impact of the microbiota could only be investigated using germ-free mice and gnotobiotic technology. To avoid the use of a chronic injury model (DSS), our laboratory has substituted the spontaneous IL-10−/− mouse model of intestinal inflammation for the AOM/DSS model. We have focused our attention on the role of microbial community on the development of colitis-associated colorectal cancer. Using this AOM/Il10−/− model of colorectal cancer in combination with gnotobiotic techniques, we observed a clear role for the microbiota in the development and progression of the pathology. Using metagenomic approaches, we observed a shift in bacterial community composition in mice bearing tumors compared to healthy controls. Interestingly, introduction of the probiotic VSL#3 changed microbial community structure and promoted the development of colorectal cancer in Il10−/− mice. Gnotobiotic experiments showed that bacteria-induced intestinal inflammation is not sufficient to promote colorectal cancer. Indeed, although the severity of colitis at the clinical and molecular levels is similar between Escherichia coli NC101 (E. coli) and Enterococcus faecalis (E. faecalis) monoassociated Il10−/− mice, tumor numbers were enhanced by more than 40 fold by the former commensal bacterium. We will present evidence that microbial composition and its associated genome/transcriptome plays a key role in the development of CAC. These findings would suggest that although chronic inflammation is a part of the pathology, microbial community and associated microbial genome is at the forefront of CAC susceptibility. Altering the composition of the microbiota may represent a novel means by which to modulate development of colorectal cancer in IBD patients. Citation Information: Cancer Prev Res 2010;3(12 Suppl):ED06-04.

An inducible mouse model of colon carcinogenesis for the analysis of sporadic and inflammation-driven tumor progression

Colorectal cancer is a life-threatening disease that can develop spontaneously or as a complication of inflammatory bowel diseases. Mouse models are essential tools for the preclinical testing of novel therapeutic options in vivo. Here, we provide a highly reliable protocol for an experimental mouse model to study the development of colon cancers. It is based on the mutagenic agent azoxymethane (AOM), which exerts colonotropic carcinogenicity. Repeated intraperitoneal administration of AOM results in the development of spontaneous tumors within 30 weeks. As an alternative option, inflammation-dependent tumor growth can be investigated by combining the administration of AOM with the inflammatory agent dextran sodium sulfate in drinking water, which causes rapid growth of multiple colon tumors per mouse within 10 weeks. Different scoring systems including number of tumors and tumor size identify factors promoting or inhibiting tumor initiation and/or tumor progression, respectively.

Stiffness and Fraction of Myosin Motors Responsible for Active Force in Permeabilized Muscle Fibers from Rabbit Psoas

The stiffness of the single myosin motor ( ɛ) is determined in skinned fibers from rabbit psoas muscle by both mechanical and thermodynamic approaches. Changes in the elastic strain of the half-sarcomere (hs) are measured by fast mechanics both in rigor, when all myosin heads are attached, and during active contraction, with the isometric force ( T 0) modulated by changing either [Ca 2+] or temperature. The hs compliance is 43.0 ± 0.8 nm MPa −1 in isometric contraction at saturating [Ca 2+], whereas in rigor it is 28.2 ± 1.1 nm MPa −1. The equivalent compliance of myofilaments is 21.0 ± 3.3 nm MPa −1. Accordingly, the stiffness of the ensemble of myosin heads attached in the hs is 45.5 ± 1.7 kPa nm −1 in isometric contraction at saturating [Ca 2+] ( e 0), and in rigor ( e r) it rises to 138.9 ± 21.2 kPa nm −1. ɛ, calculated from e r and the lattice molecular dimensions, is 1.21 ± 0.18 pN nm −1. ɛ estimated, using a thermodynamic approach, from the relation of T 0 at saturating [Ca 2+] versus the reciprocal of absolute temperature is 1.25 ± 0.14 pN nm −1, similar to that estimated for fibers in rigor. Consequently, the ratio e 0/ e r (0.33 ± 0.05) can be used to estimate the fraction of attached heads during isometric contraction at saturating [Ca 2+]. If the osmotic agent dextran T-500 (4 g/100 ml) is used to reduce the lateral filament spacing of the relaxed fiber to the value before skinning, both e 0 and e r increase by ∼40%. ɛ becomes ∼1.7 pN nm −1 and the fraction and the force of myosin heads attached in the isometric contraction remain the same as before dextran application. The finding that the fraction of myosin heads attached to actin in an isometric contraction is 0.33 rules out the hypothesis of multiple mechanical cycles per ATP hydrolyzed.

Inhibition of Coreceptor-Independent Cell-to-Cell Human Immunodeficiency Virus Type 1 Transmission by a CD4-Immunoglobulin G2 Fusion Protein

We have previously shown (J. Blanco et al., J. Biol. Chem. 279:51305-51314, 2004) that the contact between HIV producing cells and primary CD4(+) T cells may induce the uptake of human immunodeficiency virus (HIV) particles by target cells in the absence of HIV envelope-mediated membrane fusion or productive HIV replication. HIV uptake by CD4(+) T cells was dependent on cellular contacts mediated by the binding of gp120 to CD4 but was independent of the expression of the appropriate HIV coreceptor, CCR5 or CXCR4. Here, we have characterized the effect of agents blocking gp120 binding to CD4 on cell-to-cell HIV transmission. A recombinant CD4-based protein (CD4-immunoglobulin G2 [IgG2]), that is currently being evaluated in clinical trials, completely inhibited the uptake of HIV particles by CD4(+) T cells from persistently infected cells expressing R5, X4, or X4/T-20-resistant HIV-1 envelope glycoproteins. Consequently, both the release of viral particles from endocytic vesicles and the infection of reporter U87-CD4 cells were also prevented. The polyanionic anti-HIV agent dextran sulfate failed to prevent the intracellular uptake of virions by CD4(+) T cells. Indeed, it increased HIV uptake in a dose-dependent manner, suggesting functional differences between the specific gp120-targeting CD4-IgG2 agent and nonspecific HIV binding inhibitors. Thus, the inhibition of the specific interaction between gp120 and CD4 protein could be an effective strategy to inhibit HIV binding to CD4(+) T cells, and the mechanism by which CD4(+) T cells lacking the appropriate coreceptor may be converted in HIV carriers.

Confinement and crowding effects on tryptophan synthase α 2β 2 complex

Biological molecules experience in vivo a highly crowded environment. The investigation of the functional properties of the tryptophan synthase α 2β 2 complex either entrapped in wet nanoporous silica gels or in the presence of the crowding agents dextran 70 and ficoll 70 indicates that the rates of the conformational transitions associated to catalysis and regulation are reduced, and an open and less catalytically active conformation is stabilized.

Effect of dextran-induced changes in refractive index and aggregation on optical properties of whole blood

The purpose of the present study is to investigate systematically the mechanisms of alterations in the optical properties of whole blood immersed in the biocompatible agent dextran, and to define the optimal concentration of dextrans required for blood optical clearing in order to enhance the capability of light penetration depth for optical imaging applications. In the experiments, dextrans with different molecular weights and various concentrations were employed and investigated by the use of the optical coherence tomography technique. Changes in light attenuation, refractive index and aggregation properties of blood immersed in dextrans were studied. It was concluded from the results that the mechanisms for blood optical clearing are characteristic of the types of dextrans employed, their concentrations and the application stages. Among the substances applied, Dx500 at a concentration at 0.5 g dl−1 gives the best result in improving light penetration depth through the blood. The increase of light transmission at the beginning of the addition of dextrans is mainly attributed to refractive index matching between the scattering centres and the ground matter. Thereafter, the transmission change is probably due to a dextran-induced aggregation–disaggregation effect. Overall, light scattering in the blood could be effectively reduced by the application of dextrans. It represents a promising approach to increasing the imaging depth for in vivo optical imaging of biological tissue, for example optical coherence tomography.

Corneal Cryopreservation with Dextran

Different methods of corneal cryopreservation have been introduced, those employing intracellular cryoprotectants such as Me2SO or glycerol being the most widely favored. We investigated the influence of several freeze-thaw trauma variables on the survival of porcine endothelial monolayers when employing the extracellular cryoprotective agent dextran. We first examined the effects of various dextran concentrations and then, having ascertained the optimal concentration, further investigated the influence of fetal calf serum (FCS) concentration in the cryopreservation medium, the cooling rate, the thawing temperature, and the length of the preincubation in the freezing medium prior to cryopreservation. The numerical densities of endothelial cells were determined at dissection in hypoosmotic balanced salt solution and after organ culture by staining with alizarin red S and trypan blue. Morphological evaluation was not performed directly after thawing but after a subsequent organ culture at 37°C to detect latent cell damage after freeze-thaw trauma. Our data revealed that corneas cryopreserved in minimal essential medium containing 10% dextran but lacking FCS, preincubated for 3 h, frozen at a cooling rate of 1°C/min, and thawed at 37°C incurred the lowest cell losses (22.4%, SD ± 3.8). We conclude that dextran is an effective cryoprotectant for freezing of porcine corneas. However, variations between species in the results of cryopreservation require further investigation of an in vivo animal model and studies with human corneas before its clinical use can be recommended.

Iodinated radiographic contrast media inhibit shear stress- and agonist-evoked release of NO by the endothelium.

1 We have used isolated arterial preparations from the rabbit and dog to investigate whether non-ionic iodinated radiographic contrast media (IRCM) modulate nitric oxide (NO) release. The tri-iodinated monomers iopromide and iohexol were compared with the hexa-iodinated dimer iodixanol. 2 The vasodilator effects of iohexol (300 mg ml-1) and iodixanol (320 mg ml-1) were assessed in cascade bioassay. Increasing concentrations of iohexol or iodixanol caused concentration-dependent relaxations of the detector tissue which were insensitive to 100 microM NG-nitro L-arginine methyl ester (L-NAME) and 10 microM indomethacin, whereas viscosity-associated relaxations induced by the 'inert' agent dextran (MW 80,000; 1-4%) were attenuated by inhibition of NO synthesis. 3 Relaxations of endothelium-intact rings to acetylcholine (ACh) were attenuated by preincubation with iohexol or iodixanol, whereas relaxations to sodium nitroprusside (SNP) in endothelium-denuded rings were unaffected. Inhibitory activity did not correlate with either molarity or iodine concentration. Mannitol caused inhibition of both ACh- and SNP-induced responses. 4 In isolated perfused arteries the depressor responses to iodixanol (320 mg ml-1) and iopromide (300 mg ml-1) administered as close arterial bolus attained a plateau with maximal dilatations of approximately 25% and approximately 60%, respectively. Addition of 100 microM NG-nitro L-arginine (L-NOARG) and/or 10 microM indomethacin to the perfusate had no effect on the responses to either agent. 5 We conclude that IRCM exert direct effects on the endothelium that inhibit NO production rather than its action on vascular smooth muscle. Shear stress-induced stimulation of NO production by IRCM is unlikely to contribute to their vasodilator activity in vivo when administered during angiography despite high intrinsic viscosity.

Effect of osmotic solutes on human mesothelial cell proliferation in vitro.

We evaluated the acute effects of various solutions with high osmotic concentrations on the proliferation of human peritoneal mesothelial cells (HPMC) in vitro culture. HPMC proliferation was assessed by the 3Hthymidine incorporation. Damage to the HPMC membrane was measured by augmented release of the cytosolic enzyme lactic dehydrogenase (LDH). The various osmotic solutions included the crystalloid agents (glucose, raffinose, sorbitol, sucrose, mannitol, glycerol, a mixture of essential amino acids) at 50mM or 100mM and oncotic agents (dextran 70, maltodextrin, hydroxyethylstarch 20) at 3.5% or 7%.All of the solutions with high osmotic concentrations at neutral pH inhibited the growth of HPMC in a concentration-dependent way. The inhibition by the crystalloid agents was stronger than that by the oncotic agents. The amino acids caused very strong inhibition of HPMC growth. At the higher concentration (100mM), most of crystalloid agents also damaged the HPMC membrane, but no significant injury to HPMC was observed at the higher concentration (7%) of the oncotic agents.The results suggest that the toxic effect of the osmotic solutes on the proliferation of HPMC depends on the hyperosmolality. However, we cannot exclude a toxic metabolic effect on the cells. The oncotic solutes seem to be less toxic for mesothelial cell proliferation.

T30177, an oligonucleotide stabilized by an intramolecular guanosine octet, is a potent inhibitor of laboratory strains and clinical isolates of human immunodeficiency virus type 1.

T30177, an oligonucleotide composed of only deoxyguanosine and thymidine, is 17 nucleotides in length and contains single phosphorothioate internucleoside linkages at its 5' and 3' ends for stability. This oligonucleotide does not share significant primary sequence homology with or possess any complementary (antisense) sequence motifs to the human immunodeficiency virus type 1 (HIV-1) genome. T30177 inhibited replication of multiple laboratory strains of HIV-1 in human T-cell lines, peripheral blood lymphocytes, and macrophages. T30177 was also found to be capable of inhibiting multiple clinical isolates of HIV-1 and preventing the cytopathic effect of HIV-1 in primary CD4+ T lymphocytes. In assays with human peripheral blood lymphocytes there was no observable toxicity associated with T30177 at the highest concentration tested (100 microM), while the median inhibitory concentration was determined to be in the range of 0.1 to 1.0 microM for the clinical isolates tested, resulting in a high therapeutic index for this drug. In temporal studies, the kinetics of addition of T30177 to infected cell cultures indicated that, like the known viral adsorption blocking agents dextran sulfate and Chicago sky blue, T30177 needed to be added to cells during or very soon after viral infection. However, analysis of nucleic acids extracted at 12 h postinfection from cells treated with T30177 at the time of virus infection established the presence of unintegrated viral cDNA, including circular proviral DNA, in the treated cells. In vitro analysis of viral enzymes revealed that T30177 was a potent inhibitor of HIV-1 integrase, reducing enzymatic activity by 50% at concentrations in the range of 0.050 to 0.09 microM. T30177 was also able to inhibit viral reverse transcriptase activity; however, the 50% inhibitory value obtained was in the range of 1 to 10 microM, depending on the template used in the enzymatic assay. No observable inhibition of viral protease was detected at the highest concentration of T30177 used (10 microM). In experiments in which T30177 was removed from infected cell cultures at 4 days post-HIV-1 infection, total suppression of virus production was observed for more than 27 days. PCR analysis of DNA extracted from cells treated in this fashion was unable to detect the presence of viral DNA 11 days after removal of the drug from the infected cell cultures. The ability of T30177 to inhibit both laboratory and clinical isolates of HIV-1 and the experimental data which suggest that T30177 represents a novel class of integrase inhibitors indicate that this compound is a viable candidate for evaluation as a therapeutic agent against HIV-1 in humans.

Thrombotic and Hemorrhagic Complications of Stenting Coronary Arteries: Incidence, Management, and Prevention.

Stents are intravascular prostheses which provide endoluminal scaffolding, effectively reducing elastic recoil and sealing local dissections. Stents have become the treatment of choice for acute vessel closure following percutaneous coronary intervention. In addition, by providing a large, smooth lumen, stenting increases procedural success and decreases late restenosis compared with conventional balloon angioplasty. All stents evaluated clinically are constructed of non-corrosive metallic alloys which are inherently thrombogenic.The incidence of stent thrombosis ranges from 0.4-18%. While acute thrombosis is uncommon, subacute thrombosis may occur from 5 to 21 days (mean 7 days) after placement. Predictors of stent thrombosis include stenting for bailout indication, angiographically visible thrombus after implantation, stenting of smaller vessels, presence of residual dissection after stenting, poor distal runoff, incomplete stent expansion and stenting in the setting of acute myocardial infarction. Stent thrombosis is associated with high incidence of Q-wave myocardial infarction (70-90%) and mortality (7-20%), and is best treated with emergency catheterization and balloon angioplasty.To prevent stent thrombosis, aggressive procedural and postprocedural pharmacological regimens employing antiplatelet agents (aspirin, dipyridamole and dextran) and anticoagulation (heparin followed by warfarin) have been used. While these regimens have reduced the incidence of stent thrombosis to <5%, they are associated with a high incidence of vascular and hemorrhagic complications, increased length of hospitalization and total cost. To decrease the incidence of stent thrombosis and obviate the need for anticoagulation, strategies such as intravascular ultrasound guided "optimal stenting" and addition of the antiplatelet agent ticlopidine, are being evaluated. In the future coating of stents with agents such as heparin, may further reduce the risk of thrombosis and the requirement for long-term anticoagulation.

Detection and quantitation in rat tissues of the superparamagnetic magnetic resonance contrast agent dextran magnetite as demonstrated by electron spin resonance spectroscopy.

The compound studies in this article is a superparamagnetic macromolecular complex of magnetite cores coated with hydrophilic dextran, which is under active investigation as a contrast agent for magnetic resonance imaging (MRI) in liver and spleen. The biodistribution of paramagnetic compounds is problematic and is usually studied by histochemical reactions or by radiolabeling the compound under study. The purpose of this article is to show how electron spin resonance (ESR) spectroscopy detects dextran magnetite (DM) particles in tissues. DM injected intravenously in the experimental animal was detected in some reticulo-endothelial organs by ESR. The spectroscopic study was validated using electron microscopy and electron-probe microanalysis. DM exhibits an ESR spectrum; ESR delineated the distribution of DM distribution in liver, spleen, bone marrow, and blood as a function of time. The blood clearance was biphasic, dependent on the size of particles. ESR spectroscopy is a highly sensitive and reproducible method of studying DM distribution.

Approaches to improve the association of amikacin sulphate to poly(alkylcyanoacrylate) nanoparticles

Abstract Owing to the low rate of association of polar drugs, such as amikacin sulphate, to hydrophobic carriers, e.g. poly(alkylcyanoacrylate) nanoparticles, several procedures have been investigated to improve the drug carrier capacity. The greatest improvement was noted with the covalent reaction between the drug and the suspension agent dextran 70, attached to the particle surface. The antibacterial activity in this case was dramatically reduced, however. The addition of sodium lauryl sulphate to the polymerisation medium reduced the hydrosolubility of the drug, improving its incorporation into the nanoparticles. The study of the sorption behaviour of amikacin sulphate onto freeze-dried nanoparticles, prepared either using a surfactant (Synperonic F 68) or a suspension agent (dextran 70), shows preferrable results in the former case. The presence of drug in the polymerisation medium drastically increased the mean particle size, destabilising the nanoparticles at the highest concentration assayed (20 mg/ml). Less drug was adsorbed onto these particles at a later stage, which seems to be due to their larger size compared to non-loaded nanoparticles. Moreover, the drug-polymer association mechanism, investigated by zeta-potential studies, appears to be an electrostatic interaction, in keeping with the observed reduction of particle surface charge after adsorption of amikacin. Finally, the modification of the polymer molecular weight in the formulations developed was investigated.

Inflammatory particles stimulate thromboplastin production by human monocytes

Several inflammatory particles (zymosan, two forms of asbestos, carrageenan) and the soluble inflammatory agent dextran sulphate induced a dose- and time-dependent increase in thromboplastin activity of cultured human monocytes. Two weakly inflammatory particles (latex and anatase) had little effect. The increase largely depended on protein synthesis; was slow, reaching a peak at about 18 h; and was not due to endotoxin. This procoagulant may contribute to the fibrin deposition frequently seen in inflammatory lesions.