Entrapment and release kinetics study of dyes from BSA microspheres forming a matrix and a reservoir system.

Two kinds of Bovine Serum Albumin (BSA)-loaded microspheres were prepared in water-organic bilayer systems using ultrasonic irradiation. The first method included an aqueous solution of BSA and water-soluble dye together, mixed with dodecane, that upon sonication formed a matrix system where the dye is concentrated in the protein shell. The other system included an aqueous solution of BSA mixed with octanol-soluble dye that, upon sonication, formed a reservoir system in which the dye filled the inner volume of the microspheres. Each of these microspheres was prepared with two different dyes and their leaching profiles into pure solvents were studied using UV-vis spectrometry. Fast leaching was observed at the beginning for both systems, which levelled-off after a certain time. For the matrix system, an equilibrium state was obtained after 100-200 hours, whereas for the reservoir system, leaching occurred much faster, within 1-3 hours. Such systems can serve as models for drug delivery agents.

Dye-binding assays for evaluation of the effects of small molecule inhibitors on amyloid (aß) self-assembly.

Dye-binding assays, such as those utilizing Congo red and thioflavin T, are among the most widely used tools to probe the aggregation of amyloidogenic biomolecules and for the evaluation of small molecule inhibitors of amyloid aggregation and fibrillization. A number of recent reports have indicated that these dye-binding assays could be prone to false positive effects when assessing inhibitors' potential toward Aß peptides, species involved in Alzheimer's disease. Specifically, this review focuses on the application of thioflavin T for determining the efficiency of small molecule inhibitors of Aß aggregation and addresses potential reasons that might be associated with the false positive effects in an effort to increase reliability of dye-binding assays.

Detection of Aß plaques by a novel specific MRI probe precursor CR-BSA-(Gd-DTPA)n in APP/PS1 transgenic mice.

Amyloid plaques are one of the hallmarks of Alzheimer's disease (AD). The lack of specific probes that can detect individual senile plaques in AD has prompted the development of magnetic resonance imaging (MRI) probes. In this study, based on DTPA-gadolinium (III) and congo red (CR), a novel specific MRI probe precursor CR-BSA-(Gd-DTPA)n was successfully synthesized. Its ability to bind to amyloid plaques was evaluated by brain sections from APP/PS1 transgenic mice. Its specificity for Aß plaques was further demonstrated by immunohistochemistry (IHC) staining with the monoclonal antibody to the Aß protein. Meanwhile, the amyloid deposits detected by the CR-BSA-(Gd-DTPA)n were matched to the amyloid deposits detected by Aß specific antibody. We also found that a few amyloid-like deposits which was not detected by IHC. The findings indicated that the probe perhaps could detect the neurofibrillary tangles (NFT) similar to the effect of CR itself, and this will be verified in future experiments. The works suggested that the Aß protein-specific magnetic resonance contrast agent precursor CR-BSA-(Gd-DTPA)n can be used as a potential fluorescence and MR multi-modal imaging probe precursor to display individual senile plaques in AD.

In vivo accumulation of self-assembling dye Congo red in an area marked by specific immune complexes: possible relevance to chemotherapy.

Supramolecular micellar structures have been proposed as carriers in aim-oriented drug transportation to a target marked by specific immune complexes. In this study, the self-assembling dye Congo red was used as a model supramolecular carrier and its accumulation in the target was studied in vivo. The target was created in vivo as the local specific inflammation provoked by subcutaneous injection of antigen to the ear of a previously immunized rabbit. The color caused by accumulation of Congo red after its intravenous injection was registered by pictures of the ear with suitably filtered visible light shining through it to distinguish Congo red against the background color of hemoglobin. The results confirmed the expected accumulation and retention of Congo red in the inflammation area marked by deposits of specific immune complexes. The role of albumin and its possible interference with transportation of drugs through the blood by supramolecular carriers was also subjected to preliminary examination. The results revealed that albumin collaborates rather than interferes with drug transportation; this is another factor making the use of supramolecular carriers for aim-oriented chemotherapy highly promising.

Pharmacokinetics and distribution of sodium 3,4-diaminonaphthalene-1-sulfonate, a Congo Red derivative active in inhibiting PrP(res) replication.

Sodium 3,4-diaminonaphthalene-1-sulfonate (CRA) is a compound, synthesised by our group from Congo Red (CR), that is active in preventing the pathological conversion of normal prion protein (PrP). As the precise mechanisms controlling the ways in which prions are distributed and infect the brain and other organs are not fully understood, studying the pharmacokinetics of drugs that are active against prions may clarify their targets and their means of inhibiting prion infection. This paper describes the pharmacokinetics of CRA in plasma, spleen and brain after single or repeated intraperitoneal or subcutaneous administration, as determined by means of specific and sensitive fluorimetric HPLC. A single intraperitoneal administration led to peak plasma CRA concentrations after 15 min, followed by biphasic decay with an apparent half-life of 4.3 h. After subcutaneous administration, T(max) was reached after 30 min, and was followed by a similar process of decay: Cmax and the AUC0-last were 25% those recorded after intraperitoneal administration. The mean peak concentrations and AUCs of CRA after a single intraperitoneal or subcutaneous administration in peripheral tissue (spleen) were similar to those observed in blood, whereas brain concentrations were about 2% those in plasma. After repeated intraperitoneal or subcutaneous doses, the Cmax values in plasma, brain and spleen were similar to those observed at the same times after a single dose. After repeated intraperitoneal doses, CRA was also found in the ventricular cerebrospinal fluid at concentrations of 1.8 +/- 0.2 microg(-1) mL, which is similar to, or slightly higher than, those found in brain. Brain concentrations may be sufficient to explain the activity of CRA on PrP reproduction in the CNS. However, peripheral involvement cannot be excluded because the effects of CRA are more pronounced after intraperitoneal than after intracerebral infection.

Congo red binding test in enteroinvasive and nonpathogenic Escherichia coli strains.

Out of 6 variants the appropriate media to perform Congo red binding test for enteroinvasive E. coli strains were established (trypto-soy agar Eiken, T.S.A.--Cantacuzino Institute and B.T.S.D.). 12 E. coli strains belonging to enteroinvasive O-serogroups formed on Congo red agar red-coloured, non-coloured colonies or both; cultures from 59 red colonies and 61 white colonies were inoculated in guinea pig eyes. The correlation between positive Congo red binding test and positive Sereny test was 91% (out of 59 red colonies, 47 evoked keratoconjunctivitis in both infected eyes and 7 in only one eye). The negative Congo red binding test corresponds (98.4%) to the failure to induce illness in the guinea pigs' eye (only one out of 61 Crb = colonies was Sereny positive, evoking keratoconjunctivitis in only one of the two infected eyes of a guinea pig). Comparing in vivo lack of pathogenicity in 44 E. coli strains isolated from human normal intestinal flora and negative Congo red binding test, a correlation of 72.73% on B.T.S.D. and 65.91% on T.S.A. medium was found. Developing an appropriate method based on Crb test about 70% of the nonpathogenic E. coli colonies could be eliminated from the laborious agglutination with enteroinvasive O-serogroups E. coli antisera.

Correlation between Congo red binding as virulence marker in Shigella species and Sereny test.

Six variants of nutrient agar were tested in order to chose the suitable media for Congo red binding test. Trypto-soy Eiken, T.S.A - Cantacuzino Institute and B.T.S.D. (a medium prepared with Difco ingredients) are appropriate to distinguish between virulent Crb+ and avirulent Crb- strains. Congo red binding was compared with Sereny test using 25 Shigella strains. The strains were inoculated onto trypto-soy agar Eiken plates with 0.01% Congo red, incubated 24 hours at 37 degrees C. A number of each kind (Crb+ and Crb-) of colonies developed by every strain was subcultured on nutrient agar and Sereny test was performed with these cultures. As expected, all 84 Crb+ colonies in vivo tested, produced keratoconjunctivitis. In the case of Crb- colonies a proper correlation with Sereny negative test was observed in 57 out of 73 colonies (78.2%) to which 10.9% (8 out of 73) less virulent (evoking illness in only one of the two inoculated eyes) colonies may be added. As our results confirmed that loss of pigmentation was consistently accompanied by loss or diminishing of virulence, we consider that Congo red binding may be used as an alternative of in vivo test for establishing the virulence of Shigellae in the routine practice of microbiology laboratories which usually are not provided with cell cultures or animals. Its reduced cost is an important advantage, too.

[The antiinflammatory and immunostimulating activities of S-4001--a polysaccharide isolated from Lei Wan (Polyporus mylitiae)].

Lei Wan, Poliporus mylittae Cook et Mass (Omphalia lapidescens Schraet) is a kind of fungus used in traditional Chinese medicine, as an anthelminthic. From Lei Wan, an active component designated as S-4001 had been isolated. Preliminary results indicate that S-4001 belongs to D, beta, 1-3 glucan with some 1-6 linkages. After administration of S-4001, significant antiinflammatory activity was found in various experimental animal models, including croton oil induced ear edema in mice and agar or yeast induced ankle swelling in rats. An inhibitory action on leucocyte migration induced by intraperitoneal injection of CMC in rats was also observed. The plasma content of corticosterone was significantly increased, but the content of ascorbic acid in the adrenals did not change in rats given S-4001. Apart from these actions, S-4001 showed a number of immunostimulating actions such as increasing the clearance of Congo red from mice blood and potentiating the immunohemolysis reaction in 615 mice.

The formation of soluble heat IgG aggregates for immunological studies.

The structural alteration associated with the aggregation of heated immunoglobulin molecules and the triggering of their effector reactivity was studied using soluble aggregate derivatives. Large but still soluble aggregate species were obtained by the formation of complexes of heated immunoglobulin molecules with azo dyes - Congo Red or Trypan Blue. Exposed peptide fragments in the area rearranged on heating were identified by short-lasting proteolysis. The altered area was in this way localized in the Fab portion of the heavy chain. The heavy chain fragment bordered by disulphides which stabilize the domains V and CH1 was indicated as a most probable peptide portion forming the link between the aggregating molecules. The obtained dye-aggregate species retain their effector activity. The effect of structural alterations in the Fab fragment on the Fc remains unclear.