Low and high molecular weight chitosans interactions with Streptococcus mutans: an in vitro study.

We evaluated the in vitro capacity of high and low molecular weight chitosans (HMWCh and LMWCh) to inhibit the adherence of strains of S. mutans obtained from the American Type Culture Collection (ATCC,25175) to artificial saliva-coated hydroxiapatite beads. The effect of these biopolymers was assessed in terms of pH, ionic force, minimum inhibitory concentration (MIC) and antibacterial activity. The results show that HMWCh is modified by a rise in pH (7.0) and ionic strength. The induced conformational changes lead to the formation of rigid meshes capable of aggregating and entrapping S. mutans. This process is associated to the properties of HMWCh. LMWCh gave rise to smaller aggregates that exhibited a comparatively reduced interaction capacity. The MIC for HMWCh was 0.5 g% and evidenced the bacteriostatic action of the aggregates. We conclude that HMWCh would exert an inhibitory effect on the process of specific adsorption of S. mutans to saliva-coated hydroxiapatite beads.

Low and high molecular weight chitosans interactions with Streptococcus mutans: an in vitro study.

We evaluated the in vitro capacity of high and low molecular weight chitosans (HMWCh and LMWCh) to inhibit the adherence of strains of S. mutans obtained from the American Type Culture Collection (ATCC,25175) to artificial saliva-coated hydroxiapatite beads. The effect of these biopolymers was assessed in terms of pH, ionic force, minimum inhibitory concentration (MIC) and antibacterial activity. The results show that HMWCh is modified by a rise in pH (7.0) and ionic strength. The induced conformational changes lead to the formation of rigid meshes capable of aggregating and entrapping S. mutans. This process is associated to the properties of HMWCh. LMWCh gave rise to smaller aggregates that exhibited a comparatively reduced interaction capacity. The MIC for HMWCh was 0.5 g

and evidenced the bacteriostatic action of the aggregates. We conclude that HMWCh would exert an inhibitory effect on the process of specific adsorption of S. mutans to saliva-coated hydroxiapatite beads.

Low and high molecular weight chitosans interactions with Streptococcus mutans: an in vitro study

We evaluated the in vitro capacity of high and low molecular weight chitosans (HMWCh and LMWCh) to inhibit the adherence of strains of S. mutans obtained from the American Type Culture Collection (ATCC,25175) to artificial saliva-coated hydroxiapatite beads. The effect of these biopolymers was assessed in terms of pH, ionic force, minimum inhibitory concentration (MIC) and antibacterial activity. The results show that HMWCh is modified by a rise in pH (7.0) and ionic strength. The induced conformational changes lead to the formation of rigid meshes capable of aggregating and entrapping S. mutans. This process is associated to the properties of HMWCh. LMWCh gave rise to smaller aggregates that exhibited a comparatively reduced interaction capacity. The MIC for HMWCh was 0.5 g

and evidenced the bacteriostatic action of the aggregates. We conclude that HMWCh would exert an inhibitory effect on the process of specific adsorption of S. mutans to saliva-coated hydroxiapatite beads.

A comparative study in vivo of the therapeutic effect of triclosan, hexetidine and chitosan.

The present clinical study was performed to comparatively assess the therapeutic effect of Low and High Molecular Weight Chitosan (LMWCh and HMWCh), hexetidine, triclosan. Plaque index, saliva buffering capacity and bacteriological controls for S. mutans and lactobacilli, were performed. The plaque and bacterial indices revealed statistically significant differences between groups. Buffering capacity was similar using, hexetidine, and triclosan, whereas it was maximum in 100% of the patients in the LMWCh and HMWCh groups. Only 0.5% HMWCh induced low activity of S. mutans in 100% of the patients and caused complete inhibition of lactobacilli growth. No changes were observed in the profile of salivary proteins. The present clinical study confirms the therapeutic efficacy of the chitosan as a bacteriostatic agent.

A comparative study in vivo of the therapeutic effect of triclosan, hexetidine and chitosan.

The present clinical study was performed to comparatively assess the therapeutic effect of Low and High Molecular Weight Chitosan (LMWCh and HMWCh), hexetidine, triclosan. Plaque index, saliva buffering capacity and bacteriological controls for S. mutans and lactobacilli, were performed. The plaque and bacterial indices revealed statistically significant differences between groups. Buffering capacity was similar using, hexetidine, and triclosan, whereas it was maximum in 100

of the patients in the LMWCh and HMWCh groups. Only 0.5

HMWCh induced low activity of S. mutans in 100

of the patients and caused complete inhibition of lactobacilli growth. No changes were observed in the profile of salivary proteins. The present clinical study confirms the therapeutic efficacy of the chitosan as a bacteriostatic agent.

A comparative study in vivo of the therapeutic effect of triclosan, hexetidine and chitosan

The present clinical study was performed to comparatively assess the therapeutic effect of Low and High Molecular Weight Chitosan (LMWCh and HMWCh), hexetidine, triclosan. Plaque index, saliva buffering capacity and bacteriological controls for S. mutans and lactobacilli, were performed. The plaque and bacterial indices revealed statistically significant differences between groups. Buffering capacity was similar using, hexetidine, and triclosan, whereas it was maximum in 100

of the patients in the LMWCh and HMWCh groups. Only 0.5

HMWCh induced low activity of S. mutans in 100

of the patients and caused complete inhibition of lactobacilli growth. No changes were observed in the profile of salivary proteins. The present clinical study confirms the therapeutic efficacy of the chitosan as a bacteriostatic agent.

Treatment of rheumatoid arthritis by oral administration of bovine tracheal type II collagen.

We evaluated the efficacy and safety of orally administered bovine tracheal type II collagen (CGII) in the treatment of rheumatoid arthritis (RA). Twenty RA patients received 0.5 mg/day of CGII for 12 weeks. Eighteen of them had improvements in the clinical parameters studied (swollen and tender joint counts, 15-m walking time, duration of morning stiffness, and physician's global assessment of disease activity). Anti-CGII antibodies were positive in 57% and rheumatoid factor (RF) in 71% of the patients with a short history of RA ( < or =2 years), whereas only 23% of those with long histories (>2 years) presented autoantibodies to CGII and 38% had positive RF. After the treatment, four patients showed reduced RF levels and all those with detectable serum tumor necrosis factor alpha (TNF-alpha) experienced its return to normal or levels below those at study entry. Although a placebo effect cannot be discounted, the oral administration of bovine tracheal CGII induced clinical benefits in 90% of the patients, without the side effects usually associated with treatment. This is the first study showing that feeding CGII can induce reductions in RF and TNF-alpha. The data justify further controlled studies to assess the long-term efficacy of this treatment approach.

Characterization of casein micelle precipitation by chitosans.

We have found that the addition of chitosan, a cationic polymer, on whole or skim milk produces destabilization and coagulation of casein micelles that takes place without changes in the milk pH or the stability of most whey proteins. The amount of lipids recovered in the chitosan-casein aggregates was similar or higher than that obtained with rennet or acid precipitation. Approximately 70% of milk Ca2+ (approximately 750 mg/L) was found in the chitosan-induced aggregates, which is 10 and 50% higher than the amounts observed with acid or rennet coagulations, respectively. Purified alpha, beta-, and kappa-caseins were extensively precipitated by different molecular weight chitosans at pH 6.8. The phosphate groups of caseins seem not to be relevant in this interaction because dephosphorylated alpha- and beta-caseins were equally precipitated with chitosans. Analysis by optical microscopy of the chitosan-casein complex reveals that the size of the aggregates increase as the molecular weight of chitosans increase. Hydrophobic and electrostatic interactions particpate in the association and coagulation of casein micelles with chitosans of different molecular weights. The phenomenon is observed over a broad range of temperature (4 to 70 degrees C) with a reduction in the concentration of chitosan needed to precipitate the caseins that parallels a reduction in the viscosity of the chitosan solutions. Taken together, the results indicate that the electrostatic interactions may contribute energetically to the association between the two biopolymers, but the hydrophobicity of the complex would be the key determinant in the overall energetics of the reaction.

Hydrolysis of a chitosan-induced milk aggregate by pepsin, trypsin and pancreatic lipase.

The addition of chitosan to whole milk results in dose dependent destabilization and coagulation of the casein micelles and milk fat. The present study evaluates how the presence of chitosan could affect the hydrolysis of this chitosan-induced aggregate by different gastrointestinal proteases (pepsin and trypsin) and by pancreatic lipase. The chitosan-milk aggregate was hydrolyzed by pepsin and trypsin, as evaluated by the UV absorbance of TCA-soluble peptides and by urea-PAGE. The kinetics and extent of hydrolysis were independent of the casein being soluble or aggregated. The release of soluble peptides from the aggregate was independent of the presence of chitosan. A progressive inhibition of pancreatic lipase was observed in proportion to the increase in molecular weight of the chitosan employed to induce the formation of the aggregate. Interestingly, the presence of chitosan not only affected the initial velocity of the reaction, but also reduced its extent. The results indicate that a milk aggregate induced by chitosan was very well digested by gastric and intestinal proteases independently of the molecular weight of the chitosan used, and that the aggregate could retain the lipid-lowering effect of chitosan.

Chitosan-induced phospholipase A2 activation and arachidonic acid mobilization in P388D1 macrophages.

We have found that chitosan, a polysaccharide present in fungal cell walls, is able to activate macrophages for enhanced mobilization of arachidonic acid in a dose- and time-dependent manner. Studies aimed at identifying the intracellular effector(s) implicated in chitosan-induced arachidonate release revealed the involvement of the cytosolic Group IV phospholipase A2 (PLA2), as judged by the inhibitory effect of methyl arachidonoyl fluorophosphonate but not of bromoenol lactone. Interestingly, priming of the macrophages with lipopolysaccharide renders the cells more sensitive to a subsequent stimulation with chitosan, and this enhancement is totally blocked by the secretory PLA2 inhibitor 3-(3-acetamide)-1-benzyl-2-ethylindolyl-5-oxy-propanesulfonic acid (LY311727). Collectively, the results of this work establish chitosan as a novel macrophage-activating factor that elicits AA mobilization in P388D1 macrophages by a mechanism involving the participation of two distinct phospholipases A2.

Immunocytochemical study of the distribution of a 16-kDa galectin in the chicken retina.

PURPOSE: To compare the distribution of a developmentally regulated 16-kDa galectin in the chicken retina at two different developmental stages: embryonic day 13 (ED13) and postnatal day 10 (PD10) retinas, by immunocytochemical analysis using light and transmission electron microscopy. METHODS: Semi-thin and thin sections from ED13 and PD10 retinas were incubated with the IgG fraction purified from a rabbit antiserum raised against the 16-kDa chicken galectin. After incubation with colloidal gold particle-labeled goat anti-rabbit IgGs, tissue sections were analyzed by light and transmission electron microscopy. To improve the observation by light microscopy, semi-thin immunostained sections were intensified by silver enhancement. RESULTS: In ED13 retinas a specific galectin labeling was detected in the region corresponding to the outer limiting membrane by light microscopy. This labeling seemed to be associated with the apical villi of Muller glial cells and their specialized junctions, as judged by transmission electron microscopy. In PD10 retinas, the more relevant finding revealed by light microscopy was the detection of a widespread immunostaining at the level of all retinal layers. The ultrastructural analysis indicated that the galectin labeling was detected at the cytoplasmic and nuclear compartments of Muller cells throughout the different retinal layers. Moreover, the labeling was detected in the inner limiting membrane in structures that resemble the end feet of Muller cells. The apical villi, and the specialized junctions of these glial cells, appeared more strongly stained in PD10 retinas than in ED13 retinas. Finally, highly intense labeling in a group of mitochondria localized in the inner segments of cone cells was observed. CONCLUSIONS: The present study clearly supports the idea that the subcellular distribution of the 16-kDa galectin changes during the development of the chicken retina. Morphologic changes associated with developmentally regulated expression and subcellular compartmentalization of the retinal galectin suggest that this lectin may be involved in the modulation of several processes in the visual system. Its presence in the apical villi of Miller cells may be related by modulatory functions between retina and pigment epithelium, but its presence in the cytoplasm and nucleus of these glial cells suggests a potential immunomodulatory role and its involvement in different metabolic processes between Muller and the other retinal cells. Finally, although the presence of galectins inside mitochondria has not been described before, this localization gives rise to the idea that this lectin may be involved in the modulation of mitochondrial processes.

Galectins: a key intersection between glycobiology and immunology.

Galectins are a family of evolutionarily conserved animal lectins, widely distributed from lower invertebrates to mammals. They share sequence and structure similarities in the carbohydrate recognition domain and specificity for polylactosamine-enriched glycoconjugates. In the last few years significant experimental data have been accumulated concerning their participation in different biological processes requiring carbohydrate recognition such as cell adhesion, cell growth regulation, inflammation, immunomodulation, apoptosis and metastasis. In the present review we will discuss some exciting questions and advances in galectin research, highlighting the significance of these proteins in immunological processes and their implications in biomedical research, disease diagnosis and clinical intervention. Designing novel therapeutic strategies based on carbohydrate recognition will provide answers for the treatment of autoimmune disorders, inflammatory processes, allergic reactions and tumor spreading.

Galectins: a key intersection between glycobiology and immunology

Galectins are a family of evolutionarily conserved animal lectins, widely distributed from lower invertebrates to mammals. They share sequence and structure similarities in the carbohydrate recognition domain and specificity for polylactosamine-enriched glycoconjugates. In the last few years significant experimental data have been accumulated concerning their participation in different biological processes requiring carbohydrate recognition such as cell adhesion, cell growth regulation, inflammation, immunomodulation, apoptosis and metastasis. In the present review we will discuss some exciting questions and advances in galectin research, highlighting the significance of these proteins in immunological processes and their implications in biomedical research, disease diagnosis and clinical intervention. Designing novel therapeutic strategies based on carbohydrate recognition will provide answers for the treatment of autoimmune disorders, inflammatory processes, allergic reactions and tumor spreading.

[The importance of IgG auto-antibodies, anti-collagen type II specific in Menière's disease and progressive hearing loss]. / La importancia de los auto-anticuerpos IgG, específicos anticolágeno tipo II en la enfermedad de Menière y pérdida progresiva de la audición.

UNLABELLED: The Meniere's Disease and Progressive Hearing Loss were considered idiopathic. Both entities were produced by endo lymphatic hydrops and disruption of the membrane which contain type II collagen. The inner ear presented widely expression of type II collagen. These pathologies were probably autoimmune diseases. The aim of this work was to study the relationship of specific IgG to type II collagen in Meniere's disease, Progressive hearing loss, and compared with Sudden hearing loss patients, vascular vertigo patients and normal controls. Patients were divided by clinical findings in: 1 degree Meniere's disease (n:27), 2 degrees Progressive Hearing loss (n:20), 3 degrees Sudden hearing loss (n:15), 4 degrees Vascular Vertigo (n:9) and compared with normal controls (n:30) aged and sex matched. We have measured specific IgG to type II collagen by ELISA test. We considered positive the OD two or more SD above the mean of normal controls. RESULTS: 1 degree The Meniere's group presented IgG to type II collagen (+) in 22 out of 27 patients, p < .025; 2 degrees The Progressive Hearing loss presented IgG to type II collagen in all cases (n:20), p < .0005. The Sudden Hearing loss presented IgG to type II collagen (-) in all cases (n:15) p < .00001 and Vascular Vertigo (n:9) presented IgG to type II collagen (-) in 8 out of 9 cases, p < .0005. These results suggest strongly the notion that Meniere's diseases and Progressive hearing loss have specific IgG to type II collagen and these conditions were ascribed with in autoimmune process.

La importancia de los auto-anticuerpos IgG, específicos anticolágeno tipo II en la enfermedad de MeniÞre y pérdida progresiva de la audición. / [The importance of IgG auto-antibodies, anti-collagen type II specific in MeniÞres disease and progressive hearing loss]

The Menieres Disease and Progressive Hearing Loss were considered idiopathic. Both entities were produced by endo lymphatic hydrops and disruption of the membrane which contain type II collagen. The inner ear presented widely expression of type II collagen. These pathologies were probably autoimmune diseases. The aim of this work was to study the relationship of specific IgG to type II collagen in Menieres disease, Progressive hearing loss, and compared with Sudden hearing loss patients, vascular vertigo patients and normal controls. Patients were divided by clinical findings in: 1 degree Menieres disease (n:27), 2 degrees Progressive Hearing loss (n:20), 3 degrees Sudden hearing loss (n:15), 4 degrees Vascular Vertigo (n:9) and compared with normal controls (n:30) aged and sex matched. We have measured specific IgG to type II collagen by ELISA test. We considered positive the OD two or more SD above the mean of normal controls. RESULTS: 1 degree The Menieres group presented IgG to type II collagen (+) in 22 out of 27 patients, p < .025; 2 degrees The Progressive Hearing loss presented IgG to type II collagen in all cases (n:20), p < .0005. The Sudden Hearing loss presented IgG to type II collagen (-) in all cases (n:15) p < .00001 and Vascular Vertigo (n:9) presented IgG to type II collagen (-) in 8 out of 9 cases, p < .0005. These results suggest strongly the notion that Menieres diseases and Progressive hearing loss have specific IgG to type II collagen and these conditions were ascribed with in autoimmune process.

Specific inhibition of lymphocyte proliferation and induction of apoptosis by CLL-I, a beta-galactoside-binding lectin.

Beta-galactoside-binding lectins or galectins are a family of closely related carbohydrate-binding proteins which functions still remain to be elucidated. Several evidence suggest they could play a role in different biological processes, such as cell growth regulation and immunomodulation. In the present study we report that affinity-purified CLL-I (chicken lactose lectin-I), an acidic 16-kDa galectin exhibits specific growth regulatory properties. Con A-stimulated rat spleen mononuclear cells showed a marked dose-dependent growth inhibition upon incubation with the galectin protein. Cell growth arrest was highly prevented by galectin-specific sugars. In addition, biochemical, cytofluorometrical, and morphological evidence are also provided to show that these inhibitory properties are related to a positive control in the apoptotic threshold of spleen mononuclear cells. Flow cytometric analysis showed a dose- and time-dependent increase of cells with hypodiploid DNA content upon exposure to CLL-I. Moreover, cells treated with CLL-I displayed the typical ultrastructural changes compatible with apoptosis, mainly chromatin condensation and margination along the inner surface of the nuclear envelope. Finally, the highly characteristic "ladder" pattern of DNA fragmentation into oligonucleosome-length fragments of approximately 180-200 bp could be found within 6 h of cell culture with CLL-I, mainly in the T cell-enriched population. Induction of apoptosis by a beta-galactoside-binding protein highlights a potentially novel mechanism for regulating the immune response and points to a rational basis for the postulated immunomodulatory properties of this protein family.

Regulated expression of a 16-kd galectin-like protein in activated rat macrophages.

We investigated the presence of a galectin-like protein in rat mononuclear cells using a polyclonal antibody raised against a soluble lactose-binding lectin purified from adult chicken liver that immunoreacted strongly with a broad protein band of about 16 kd in Western blot assays. Immunochemical studies revealed a constitutive expression of this protein in mononuclear cells mainly in the macrophage (M phi) population. Subcellular localization was assessed by Western blot assays of the cytosolic and membrane fractions of different cell populations studied: (1) spleen mononuclear cells, (2) T cell-enriched, (3) B cell- and M phi-enriched populations, and (4) peritoneal cells, processed in the presence of lactose. In broad agreement with immunocytochemical studies of nonpermeabilized and permeabilized cells, Western blot assays suggest that this protein is localized mainly in the cytoplasmic compartment but also associated with the cell surface. By flow cytometric analyses we detected about a 14% of ED1 double-positive cells corresponding to macrophages that constitutively express this galectin-like protein associated with their cell surface. The cytosolic fraction obtained from the M phi-enriched cell population showed hemagglutinating activity specifically inhibited by beta-galactoside-related sugars. Moreover, this galectin-like protein was retained in a lactosyl-Sepharose matrix and specifically eluted with lactose. In this work, evidence is also provided to show that different stimuli are able to modulate the expression of the galectin-like protein. Expression was upregulated in inflammatory and activated macrophages, revealing a significant increase in phorbol ester- and formylmethionine oligopeptide-treated cells. Both stimuli involving protein kinase C activation pathway have been able not only to up-regulate the total expression of this protein but also to modulate its subcellular localization.

Isolation and partial characterization of N-acetyl-D-galactosamine-binding lectins from Epiphragmophora trenquelleonis snail.

A human blood type A hemagglutinating activity was detected in albumin gland extracts of Epiphragmophora trenquelleonis snail separated by GalNAc-agarose affinity chromatography, of which two N-acetyl-D-galactosamine-binding lectins in the extracts were ETL1 was displaced from the affinity column with 1 mM GalNAc, and ETL2 with 20 mM GalNAc. Both lectins agglutinated specifically human blood type A and AB erythrocytes, but not type B and O erythrocytes. Gel filtration chromatography gave a native molecular weight of about 59 kDa for ETL1 and about 54 kDa for ETL2. On SDS-PAGE under nonreducing conditions, ETL1 showed two protein subunits of about 29 and 27 kDa, while ETL2 showed three protein subunits of about 27, 24, and 22 kDa. On SDS-PAGE under reducing conditions, both lectins showed four protein subunits of 17, 16, 12, and 11 kDa. By Western blot analyses developed with biotin-labeled lectins, N-linked oligosaccharides were detected in the 17- and 16-kDa protein subunits of ETL1 and ETL2, and in the 12-kDa protein subunit of ETL2. O-linked oligosaccharides were detected only in the 11-kDa protein subunit of ETL1 and ETL2. On isoelectric focusing both lectins exhibited microheterogeneity: ETL1 focused as three protein bands with pIs in the range of 5.6-6.0, while ETL2 focused as four protein bands with pIs in the range of 6.8-7.4. We suggest that native ETL1 and ETL2 are glycoprotein complexes with molecular weights of 59-54 kDa, composed of two 29-22-kDa nonreduced protein subunits held together by noncovalent hydrophobic interactions. Each of the nonreduced protein subunits seems to be composed of two 17-11-kDa reduced protein subunits held together by interchain disulfide linkages. The main differences between ETL1 and ETL2 could be due to different posttranslational modifications or to the relative contribution of one or more of their protein subunits.

Distribution of an endogenous 16-kd S-lac lectin in the chicken retina.

PURPOSE: To examine by indirect immunofluorescence the distribution of an endogenous 16-kd S-lac lectin (soluble lactose binding lectin) during development of the chicken retina. METHODS: Cryosections of retinal tissue at different developmental stages and cultured retinal cells (either not permeabilized or permeabilized with acetone) were incubated with a rabbit antiserum that specifically reacts with the retinal 16-kd S-lac lectin. After incubation with a fluorescent-labeled secondary antibody, tissue sections and cultured cells were analyzed by fluorescence microscopy. RESULTS: Retina was weakly stained with the antiserum on early embryonic day 7, whereas on embryonic days 13 and 18 it showed a restricted "granular" staining in the outer retina. At embryonic day 18, in addition, there was widespread staining in all retinal layers. This pattern was maintained by postnatal day 5 and in the adult retina, although the intensity of the staining of the outer retina was weaker. In retinal cell cultures, glial-like flat cells and monopolar, bipolar, and multipolar neurons were stained with the antiserum, but only if they had been previously permeabilized with acetone. CONCLUSION: The results suggest that the distribution of a 16-kd S-lac lectin changes during retinal development. Cell culture experiments indicate that most often the lectin is localized intracellularly in the different retinal cell types.

Isolation and characterization of a soluble lactose-binding lectin from postnatal chicken retina.

We investigated the presence of endogenous lectins in postnatal chicken retinal tissue assaying the hemagglutinating activity of crude soluble extracts of the tissue that was homogenized in a buffer supplemented with different sugars. Lactose was the most effective sugar to extract an hemagglutinating activity. Using similar extraction conditions, other sugars, such as glucose, N-acetylglucosamine, mannose, fucose, glucuronic and sialic acid, were ineffective to extract any significant hemagglutinating activity. The lectin was purified by affinity chromatography on lactosyl-Sepharose. SDS-PAGE and isoelectric focusing analyses showed that it has a subunit molecular weight of 16 kDa and a pI about 4.5. The retinal lectin cross-reacted immunologically with a rabbit antiserum raised against a lectin purified from adult chicken liver, which is a CLL-I (Beyer et al.: J Biol Chem 255:4236-4239, 1980) or C-16 (Sakakura et al.: J Biol Chem 265:21573-21579, 1990) form of chicken endogenous soluble lactose-binding lectins. Gel filtration studies showed that the oligomeric structure of the retinal lectin is dependent on the ionic strength of the elution buffer. The lectin hemagglutinating activity and the amount of lectin protein reached their highest levels at late developmental stages of the retinal tissue, suggesting that retinal lectin might have a functional role during terminal differentiation of retinal cells.