Analysis of Key Factors for Evaluating Mucosal Adhesion Using Swine Buccal Tissue.

The assessment of the mucoadhesive properties peak mucoadhesive force (Fmax) and work of mucoadhesion (Wmuc) with texture analyzers is a common in vitro method for analyzing formulation capabilities. Challenges arise in selecting and standardizing experimental conditions due to various variables influencing mucoadhesion. This complexity hampers direct product performance comparisons. In our study, we explored factors (contact force and time, probe speed and mucin in artificial saliva) impacting a model formulation's mucoadhesive capacity. Using Omcilon-A®Orabase on porcine buccal mucosa, we systematically varied experimental conditions, employing a statistical approach (Central Composite Design - CCD). Three variables (contact force, contact time, probe speed) and their interactions were assessed for their impact on Fmax and Wmuc. Results showed that contact time and force positively affected Fmax, while only contact time influenced Wmuc. In the mucin artificial saliva test, a force of 0.5 N, time of 600 s, and speed of 1 mm/s yielded optimal Fmax (0.587 N) and Wmuc (0.468 N.s). These conditions serve as a reference for comparing mucoadhesive properties of formulations for topical oral use.

Synthesis of Oligosaccharides Containing the S-Galp(α1 → 3)Galp Unit, Glycomimetic of the Epitope Recognized by Lytic Antibodies.

Two important activities take place in the surface of Trypanosoma cruzi, the agent of Chagas disease: the trans-sialidase (TcTS) catalyzes the transfer of sialic acid from the host glycoconjugates to the mucin-like glycoproteins from the parasite and the presence of lytic antibodies recognize the epitope α-Galp(1 → 3)-ß-Galp(1 → 4)-α-GlcNAcp. This antigenic structure is known to be present in the parasite mucins; however, in order to be substrates of trans-sialidase, some of the galactose residues should be in the ß-Galp configuration. To study the interaction between both activities, it is important to count the synthetic structures as well as the structural-related glycomimetics. With this purpose, we addressed the synthesis of a trisaccharide and two isomeric tetrasaccharides containing the 1-S-α-Galp(1 → 3)-ß-Galp motif, the thio analog of the epitope recognized by lytic antibodies. Starting with a common lactose precursor, the sulfur function was incorporated by double inversion of the configuration of the galactose residue that was further glycosylated using different activated donors. Both tetrasaccharides were good acceptors of sialic acid in the reaction catalyzed by TcTS, as determined by high-performance anion exchange chromatography.

The Glycan Structure of T. cruzi mucins Depends on the Host. Insights on the Chameleonic Galactose.

Trypanosoma cruzi, the protozoa that causes Chagas disease in humans, is transmitted by insects from the Reduviidae family. The parasite has developed the ability to change the structure of the surface molecules, depending on the host. Among them, the mucins are the most abundant glycoproteins. Structural studies have focused on the epimastigotes and metacyclic trypomastigotes that colonize the insect, and on the mammal trypomastigotes. The carbohydrate in the mucins fulfills crucial functions, the most important of which being the accepting of sialic acid from the host, a process catalyzed by the unique parasite trans-sialidase. The sialylation of the parasite influences the immune response on infection. The O-linked sugars have characteristics that differentiate them from human mucins. One of them is the linkage to the polypeptide chain by the hexosamine, GlcNAc, instead of GalNAc. The main monosaccharide in the mucins oligosaccharides is galactose, and this may be present in three configurations. Whereas ß-d-galactopyranose (ß-Galp) was found in the insect and the human stages of Trypanosoma cruzi, ß-d-galactofuranose (ß-Galf) is present only in the mucins of some strains of epimastigotes and α-d-galactopyranose (α-Galp) characterizes the mucins of the bloodstream trypomastigotes. The two last configurations confer high antigenic properties. In this review we discuss the different structures found and we pose the questions that still need investigation on the exchange of the configurations of galactose.

Multifaceted antibodies development against synthetic α-dystroglycan mucin glycopeptide as promising tools for dystroglycanopathies diagnostic.

Dystroglycanopathies are diseases characterized by progressive muscular degeneration and impairment of patient's quality of life. They are associated with altered glycosylation of the dystrophin-glycoprotein (DGC) complex components, such as α-dystroglycan (α-DG), fundamental in the structural and functional stability of the muscle fiber. The diagnosis of dystroglycanopathies is currently based on the observation of clinical manifestations, muscle biopsies and enzymatic measures, and the available monoclonal antibodies are not specific for the dystrophic hypoglycosylated muscle condition. Thus, modified α-DG mucins have been considered potential targets for the development of new diagnostic strategies toward these diseases. In this context, this work describes the synthesis of the hypoglycosylated α-DG mimetic glycopeptide NHAc-Gly-Pro-Thr-Val-Thr[αMan]-Ile-Arg-Gly-BSA (1) as a potential tool for the development of novel antibodies applicable to dystroglycanopathies diagnosis. Glycopeptide 1 was used for the development of polyclonal antibodies and recombinant monoclonal antibodies by Phage Display technology. Accordingly, polyclonal antibodies were reactive to glycopeptide 1, which enables the application of anti-glycopeptide 1 antibodies in immune reactive assays targeting hypoglycosylated α-DG. Regarding monoclonal antibodies, for the first time variable heavy (VH) and variable light (VL) immunoglobulin domains were selected by Phage Display, identified by NGS and described by in silico analysis. The best-characterized VH and VL domains were cloned, expressed in E. coli Shuffle T7 cells, and used to construct a single chain fragment variable that recognized the Glycopeptide 1 (GpαDG1 scFv). Molecular modelling of glycopeptide 1 and GpαDG1 scFv suggested that their interaction occurs through hydrogen bonds and hydrophobic contacts involving amino acids from scFv (I51, Y33, S229, Y235, and P233) and R8 and α-mannose from Glycopeptide 1.

Synthesis of the hexasaccharide from Trypanosoma cruzi mucins with the Galp(1 → 2)Galf unit constructed with a superarmed thiogalactopyranosyl donor.

Hexasaccharide ß-D-Galp-(1→ 2)-[ß-D-Galp-(1 → 3)]-ß-D-Galp-(1 → 6)-[ß-D-Galp-(1 → 2)-ß-D-Galf-(1 → 4)]-D-GlcNAc (1) was found O-linked in mucins of Trypanosoma cruzi epimastigotes and metacyclic trypomatigotes. Studies on the biological pathways and functionalities of the mucin oligosaccharides are prompted in order to understand the interactions of these molecules with the insect host. Trisaccharide constituent ß-D-Galp-(1 → 2)-ß-D-Galf-(1 → 4)-D-GlcNAc was constructed from the reducing to the non-reducing end. We discuss the difficulties to introduce a Galp unit at the O-2 position of a partially protected galactofuranosyl unit which were overcome using an anchimerically superarmed donor. By this route and employing a [3 + 3] nitrilium convergent approach hexasaccharide 1 was synthesized in moderate yield.

Synthesis of novel triazole-derived glycopeptides as analogs of α-dystroglycan mucins.

α-Dystroglycan (α-DG) mucins are essential for maintenance of the structural and functional stability of the muscle fiber and, when hypoglycosylated, they are directly involved in pathological processes such as dystroglycanopathies. Thus, this work reports the synthesis of the novel 1,2,3-triazole-derived glycosyl amino acids αGlcNAc-1-O-triazol-2Manα-ThrOH (1) and Gal-ß1,4-αGlcNAc-1-O-triazol-2Manα-ThrOH (2), followed by solid-phase assembly to get the corresponding glycopeptides NHAcThrVal[αGlcNAc-1-triazol-2Manα]ThrIleArgGlyOH (3) and NHAcThrVal[Gal-ß1,4-αGlcNAc-1-triazol-2Manα]ThrIleArgGlyOH (4) as analogs of α-DG mucins. The glycosyl amino acids 1 (72%) and 2 (35%) were synthesized by Cu(I)-assisted 1,3-dipolar azide-alkyne cycloaddition reactions (CuAAC) between the azide-glycosyl amino acid αManN3-FmocThrOBn (5) and the corresponding alkyne-functionalyzed sugars 2'-propynyl-αGlcNAc (6) and 2'-propynyl-Gal-ß1,4-αGlcNAc (7), followed by hydrogenation reactions. Subsequently, glycopeptides 3 (23%) and 4 (12%) were obtained by solid phase synthesis, involving sequential couplings of Fmoc-protected amino acids or the glycosyl amino acids 1 and 2, followed by cleavage from resin, N-acetylation and O-deacetylation (NaOMe) reactions. Lastly, enzymatic galactosylation of glycopeptide 3 with bovine ß-1,4-GalT showed that it was not a substrate for this enzyme, which could be better elucidated by docking simulations with ß-1,4-GalT.

Changes in mucins and matrix metalloproteases in the endometrium of early pregnant alpacas (Vicugna pacos).

South American Camelids (SAC) have unique reproductive features, one of which is that 98% of the pregnancies develop in the left uterine horn. Furthermore, early pregnancy is an uncharacterized process in these species, especially in regard to the ultrastructural, biochemical and genetic changes that the uterine epithelial surface undergoes to allow embryo implantation. The present study describes the uterine horn luminal surface and the characteristics of the mucinous glycocalyx in non-pregnant and early pregnant (15 days) female alpacas. In addition, the relative abundance of Mucin 1 and 16 genes (MUC1 and MUC16) was determined, as well as the relative mRNA abundance of matrix metalloproteinases (MMPs) that could be involved in MUC shedding during early pregnancy. Noticeable changes were detected in the uterine luminal epithelium and glycocalyx of pregnant alpacas in comparison to non-pregnant ones, as well as presence of MUCs and MMPs in the endometrial environment. The decrease in glycocalyx staining and in the relative abundance of MUC 1 and MUC 16 transcripts in pregnant females would allow embryo attachment to the luminal epithelium and its subsequent implantation, as has been described in other mammals. These results suggest a crucial role of MUC1 and MUC16 and a possible role of MMPs in successful embryo implantation and survival in alpacas.

Mucin and carbon nanotube-based biosensor for detection of glucose in human plasma.

This work reports an amperometric enzyme-electrode prepared with glucose oxidase, which have been immobilized by a cross-linking step with glutaraldehyde in a mixture containing albumin and a novel carbon nanotubes-mucin composite (CNT-muc). The obtained hydrogel matrix was trapped between two polycarbonate membranes and then fixed at the surface of a Pt working electrode. The developed biosensor was optimized by evaluating different compositions and the analytical properties of an enzymatic matrix with CNT-muc. Then, the performance of the resulting enzymatic matrix was evaluated for direct glucose quantification in human blood plasma. The novel CNT-muc composite provided a sensitivity of 0.44 ±â€¯0.01 mA M-1 and a response time of 28 ±â€¯2 s. These values were respectively 20% higher and 40% shorter than those obtained with a sandwich-type biosensor prepared without CNT. Additionally, CNT-muc based biosensor exhibited more than 3 orders of magnitude of linear dynamic calibration range and a detection limit of 3 µM. The short-term and long-term stabilities of the biosensors were also examined and excellent results were obtained through successive experiments performed within the first 60 days from their preparation. Finally, the storage stability was remarkable during the first 300 days.

High-resolution profiling of linear B-cell epitopes from mucin-associated surface proteins (MASPs) of Trypanosoma cruzi during human infections.

BACKGROUND: The Trypanosoma cruzi genome bears a huge family of genes and pseudogenes coding for Mucin-Associated Surface Proteins (MASPs). MASP molecules display a 'mosaic' structure, with highly conserved flanking regions and a strikingly variable central and mature domain made up of different combinations of a large repertoire of short sequence motifs. MASP molecules are highly expressed in mammal-dwelling stages of T. cruzi and may be involved in parasite-host interactions and/or in diverting the immune response. METHODS/PRINCIPLE FINDINGS: High-density microarrays composed of fully overlapped 15mer peptides spanning the entire sequences of 232 non-redundant MASPs (~25% of the total MASP content) were screened with chronic Chagasic sera. This strategy led to the identification of 86 antigenic motifs, each one likely representing a single linear B-cell epitope, which were mapped to 69 different MASPs. These motifs could be further grouped into 31 clusters of structurally- and likely antigenically-related sequences, and fully characterized. In contrast to previous reports, we show that MASP antigenic motifs are restricted to the central and mature region of MASP polypeptides, consistent with their intracellular processing. The antigenicity of these motifs displayed significant positive correlation with their genome dosage and their relative position within the MASP polypeptide. In addition, we verified the biased genetic co-occurrence of certain antigenic motifs within MASP polypeptides, compatible with proposed intra-family recombination events underlying the evolution of their coding genes. Sequences spanning 7 MASP antigenic motifs were further evaluated using distinct synthesis/display approaches and a large panel of serum samples. Overall, the serological recognition of MASP antigenic motifs exhibited a remarkable non normal distribution among the T. cruzi seropositive population, thus reducing their applicability in conventional serodiagnosis. As previously observed in in vitro and animal infection models, immune signatures supported the concurrent expression of several MASPs during human infection. CONCLUSIONS/SIGNIFICANCE: In spite of their conspicuous expression and potential roles in parasite biology, this study constitutes the first unbiased, high-resolution profiling of linear B-cell epitopes from T. cruzi MASPs during human infection.

Permeability Profiles and Intestinal Toxicity Assessment of Hydrochlorothiazide and Its Inclusion Complex with ß-Cyclodextrin Loaded into Chitosan Nanoparticles.

Here, a novel drug delivery system was developed for the hydrochlorothiazide (HCT):ß-cyclodextrin (ßCD) inclusion complex loaded into chitosan (CS) nanoparticles (NPs) [CS/HCT:ßCD NPs]. It was found, for the first time, that exposure of the intestinal mucosa to free HCT resulted in an increased and abnormal intestinal permeability associated with several injuries to the intestinal epithelium. Nevertheless, the HCT delivery system obtained ameliorated the damage of the intestinal epithelium induced by HCT. Furthermore, we found that the corresponding permeability profiles for both the free HCT and the CS/HCT:ßCD NPs were exponential and lineal, respectively. We propose that the increased intestinal uptake and severe tissue injury of HCT to the intestinal epithelium could be directly related to possible effects of this drug on the ionoregulatory Na+/K+-ATPase channel. Thus, it is postulated that the CS/HCT:ßCD NPs may increase the gastrointestinal retention of the HCT, which would provide increased adherence to the mucus barrier that lines the intestinal epithelium; consequently, this would act as a slow HCT release delivery system and maintain lower drug levels of luminal gut in comparison with the administration of free HCT, leading to less severe local injury.

Expanding the knowledge of the chemical structure of glycoconjugates from Trypanosoma cruzi TcI genotype. Contribution to taxonomic studies.

One of the main obstacles to the treatment of Chagas disease is the genetic and phenotypical variance displayed by T. cruzi strains, resulting in differences in morphology, virulence, pathogenicity and drug susceptibility. To better understand the role of glycoconjungates in Chagas disease, we performed the molecular characterization of the O-linked chains from mucins and glycoinositolphospholipids (GIPLs) of the Silvio X10 clone 1 strain. We demonstrated the presence of a ß-galactofuranose (ß-Galf) unity linked to the O-4 position of the α-N-acetylglucosamine (α-GlcNAc)O-4 in Tc-mucins. GIPLs analysis showed that the lipidic portion is exclusively composed of ceramide and the PI-oligossacharidic portion contains the Man4(AEP)GlcN-Ins-PO4 core, substituted by ethanolamine-phosphate (EtNP) on the third distal mannose from inositol, which may or may not have a terminal ß Galf unity. These results confirm the classification of the Silvio X10/1 strain in group T. cruzi I. Again, it is noted that the study of T. cruzi surface glycoconjugates confirm the molecular results and the hypothesis that surface glycoconjugates may be interesting biomarker for the differentiation of trypanosomatid strains.

Negligible elongation of mucin glycans with Gal ß1-3 units distinguishes the laminated layer of Echinococcus multilocularis from that of Echinococcus granulosus.

The larval stages of the cestodes Echinococcus multilocularis and Echinococcus granulosus cause the important zoonoses known as larval echinococcoses. These larvae are protected by a unique, massive, mucin-based structure known as the laminated layer. The mucin glycans of the E. granulosus laminated layer are core 1- or core 2-based O-glycans in which the core Galpß1-3 residue can initiate a chain comprising one to three additional Galpß1-3 residues, a motif not known in mammalian carbohydrates. This chain can be capped with a Galpα1-4 residue, and can be ramified with GlcNAcpß1-6 residues. These, as well as the GlcNAcpß1-6 residue in core 2, can be decorated with the Galpα1-4Galpß1-4 disaccharide. Here we extend our analysis to the laminated layer of E. multilocularis, showing that the non-decorated cores, together with Galpß1-3(Galpα1-4Galpß1-4GlcNAcpß1-6)GalNAc, comprise over 96% of the glycans in molar terms. This simple laminated layer glycome is exhibited by E. multilocularis grown either in vitro or in vivo. Interestingly, all the differences with the complex laminated layer glycome found in E. granulosus may be explained in terms of strongly reduced activity in E. multilocularis of a putative glycosyltransferase catalysing the elongation with Galpß1-3. Comparative inter-species analysis of available genomic and transcriptomic data suggested a candidate for this enzyme, amongst more than 20 putative (non-core 1) Gal/GlcNAc ß1-3 transferases present in each species as a result of a taeniid-specific gene expansion. The candidate gene was experimentally verified to be transcribed at much higher levels in the larva of E. granulosus than that of E. multilocularis.

A mucin-like peptide from Fasciola hepatica induces parasite-specific Th1-type cell immunity.

Fasciolosis, caused by the liver fluke Fasciola hepatica, is a major parasitic disease of livestock that causes significant economic losses worldwide. Although drugs are effective against liver flukes, they do not prevent reinfection, and continuous treatment is costly. Moreover, resistant fluke strains are emerging. In this context, vaccination is a good alternative since it provides a cost-effective long-term prevention strategy to control fasciolosis. In this paper, we evaluate the Fhmuc peptide as a potential vaccine against fasciolosis. This peptide derives from a mucin-like protein highly expressed in the infective stage of Fasciola hepatica. Mucin-like molecules expressed by parasites can contribute to several infection processes by protecting the parasite from host proteases and recognition by the immune system. We show that the Fhmuc peptide induces Th1-like immune responses specific for F. hepatica excretion-secretion products (FhESP) with a high production of IFNγ. We also investigated whether this peptide could protect animals from infection, and present preliminary data indicating that animals treated with Fhmuc exhibited reduced liver damage compared to non-immunised animals and that this protection was associated with a recruitment of B and T lymphocytes in the peritoneum, as well as eosinophils and mature dendritic cells. These results suggest that the mucin-like peptide Fhmuc could constitute a potential vaccine candidate against fasciolosis and pave the way towards the development of vaccines against parasites.

Estabelecimento de protocolos in vitro e in situ para estudos de erosão dentária / Establishment of in vitro and in situ protocols for dental erosion studies

O objetivo foi comparar uma formulação de saliva artificial com e sem mucina (in vitro) com a saliva humana (in situ) na inibição da desmineralização (subprojeto I) e no reendurecimeto de lesões de erosão (subprojeto II), e a influência do tipo de dispositivo intrabucal (mandibular X palatino) no desgaste erosivo do esmalte (subprojeto III). No subprojeto I, blocos de esmalte bovino foram selecionados pela dureza de superfície e randomizados entre os grupos: GI - saliva humana (n=30), GII - saliva artificial sem mucina (n=15), GIII - saliva artificial com mucina (n=15) e GIV - água deionizada (n=15). Quinze voluntários utilizaram o dispositivo palatino por um período 2 horas (GI). Nos grupos GII, GIII e GIV, os blocos foram imersos em suas respectivas soluções por um período de 2 horas. Imediatamente após, tanto os blocos do grupo in situ quanto dos grupos in vitro foram submetidos ao desafio erosivo inicial com ácido cítrico 1% (pH 3,6) por 4 minutos. A microdureza final foi mensurada para determinar a porcentagem de perda de dureza. No subprojeto II, os blocos, após seleção, foram erodidos in vitro e randomizados entre grupos como no subprojeto I. Para a erosão, os blocos foram imersos em ácido cítrico 1% (pH 3,6) por 4 minutos. A seguir, no grupo GI, 15 voluntários utilizaram dispositivos palatinos durante 2 horas. Nos outros grupos os blocos foram imersos nas salivas artificiais com (GIII) e sem mucina (GII) e água deionizada (GIV) por 2 horas. A precipitação de minerais sobre o esmalte foi avaliada por meio da porcentagem de recuperação de dureza. No subprojeto III, após seleção dos blocos pela dureza, os mesmos foram aleatorizados em 2 grupos (n=20): GI - dispositivo palatino e GII - dispositivo mandibular. A ciclagem consistiu na imersão dos dois dispositivos em ácido clorídrico 0,01 M (pH 2,3) por 2 minutos, 4X/dia durante 5 dias. A perda do esmalte foi avaliada por perfilometria e os voluntários responderam a um questionário quanto ao conforto dos dispositivos. Nos subprojetos I e II, os dados foram submetidos aos testes ANOVA e Tukey e no subprojeto III foi aplicado o Teste T pareado (p<0,05). Nos subprojetos I e II observou-se que todas as salivas estudadas foram capazes de promover uma recuperação de dureza do esmalte e nenhuma diferença foi encontrada entre elas (p<0,05). No ensaio de desmineralização, a saliva artificial com mucina e a saliva humana (in situ) promoveram menor perda de dureza, não mostrando diferença entre elas (p<0,05). No subprojeto III os resultados mostraram que os blocos localizados no dispositivo palatino (GI) apresentaram maior desgaste erosivo quando comparados aos do dispositivo mandibular (GII). Além disso, todos voluntários relataram maior conforto no uso do dispositivo palatino. Considerando que o dispositivo palatino é mais confortável e resultou em maior perda de esmalte quando comparado ao mandibular, sugere-se o uso de dispositivos palatinos em protocolos in situ que queiram mimetizar pacientes com alto risco de erosão dentária. Para estudos in vitro, a saliva com mucina mostrou-se como uma boa substituta à saliva humana.(AU)

The aim was to compare artificial saliva formulation with and without mucin (in vitro) with human saliva (in situ) on the inhibition of erosive demineralization (subproject I) and on the rehardening of erosion lesions (subproject II), and analyze the influence of the type of intraoral appliance (mandibular X maxillary) in enamel wear caused by erosive challenges (subproject III). In the subproject I, bovine enamel blocks were selected by initial surface hardness and randomized among the groups: GI - human saliva (n=30), GII - artificial saliva without mucin (n=15), GIII - artificial saliva with mucin (n=15) and GIV - deionized water (n=15). Fifteen volunteers wore palatal appliances for 2 hours (GI). In the GII, GIII and GIV groups, the blocks were immersed to the respective solutions for 2 hours. Subsequently, both in vitro and in situ blocks were subjected to initial erosive challenge in 1% citric acid (pH 3.6) for 4 minutes. Final enamel hardness was measured to determine the protective capacity of saliva tested by percentage of hardness loss. For the subproject II, after blocks selection, they were in vitro eroded and randomly among the groups as in subproject I. For erosion, the enamel blocks were immersed on 1% citric acid (pH 3.6) for 4 minutes. Then, in the GI, 15 volunteers wore palatal appliances for 2 hours. In the other groups the blocks were immersed in artificial saliva with (GIII) and without mucin (GII) and deionized water (GIV) for 2 hours. The minerals precipitation on the enamel was evaluated by the percentage of hardness recovery. On subproject III, after enamel blocks selection by surface hardness, they were randomly divided into 2 groups (n=20): GI - palatine appliance and GII - mandibular appliance. Erosive cycling consists in immersing of both devices in 0.01 M hydrochloric acid (pH 2.3) for 2 minutes, 4X/day during 5 days. The analysis of the wear was measured by profilometry and volunteers answered a questionnaire about the comfort of the devices. In the subprojects I and II, data were analyzed by one-way ANOVA and Tukeys test, and in the subproject III It was applied paired t-test (p <0.05). In subprojects I and II it was observed that all studied saliva promoted enamel rehardening and no difference was found between them (p <0.05). In the demineralization test, the artificial saliva with mucin and human saliva (in situ) provided lower enamel hardness loss, showing no difference between them (p<0.05). In the subproject III, the results showed that the specimens allocated in palatine appliance (GI) presented significantly higher erosive wear when compared to the specimens fixed in mandibular appliance (GII). In addition, all volunteers reported greater comfort in using the palatal device. Considering the palatal device is more comfortable and resulted in higher enamel loss when compared to the mandibular device, it is suggested the use of palatine appliances in in situ protocols who want to mimic a patient at high risk of dental erosion. For in vitro studies, the saliva with mucin might be a good substitute for human saliva.(AU)

Rheological, mucoadhesive and textural properties of thermoresponsive polymer blends for biomedical applications.

The development of binary polymeric mixtures (polymer blends) containing bioadhesive and thermoresponsive polymers can provide new materials for biomedical applications, with higher contact, increased adhesion, prolonged residence time, protection, and in determined cases, secured absorption of an active agent from the site of application. Mixtures were prepared using a wide range of poloxamer 407 and Carbopol 971P(®) amounts. The rheological (flow and oscillatory), sol-gel transition temperature, mechanical (hardness, compressibility, adhesiveness, cohesiveness and elasticity), softness, and mucoadhesive properties of formulations were investigated. Moreover, the interaction between the different proportions of polymers was also analyzed. Continuous shear and oscillatory rheometry identified the plastic flow with various degrees of thixotropy, besides the viscoelastic behavior of formulations. The determination of gelation temperature displayed values ranged from 27.17 to 41.09°C. It was also found that low carbomer concentrations were enough to provide positive interaction parameter. However, the highest values were obtained for the polymeric blends with higher concentration of poloxamer 407. The mucoadhesion and softness index were greater in preparations containing 20% (w/w) poloxamer 407. The rheological, mechanical and mucoadhesive properties of the polymeric blends can be manipulated by changing the concentrations of the polymers and they suggest the blends are worthy of biomedical applications.

Synthesis of the O-linked hexasaccharide containing ß-D-Galp-(1→2)-D-Galf in Trypanosoma cruzi mucins. Differences on sialylation by trans-sialidase of the two constituent hexasaccharides.

The hexasaccharide ß-D-Galp-(1→2)-[ß-D-Galp-(1→3)]-ß-D-Galp-(1→6)-[ß-D-Galp(1→2)-ß-D-Galf(1→4)]-D-GlcNAc (10) and its ß-D-Galf-(1→2)-ß-D-Galf containing isomer (7) are the largest carbohydrates in mucins of some strains of Trypanosoma cruzi. The terminal ß-D-Galp units are sites of sialylation by the parasite trans-sialidase. Hexasaccharide 10 was chemically synthesized for the first time by a [3+3] nitrilium based convergent approach, using the trichloroacetimidate method of glycosylation. The (1)H NMR spectrum of its alditol was identical to the spectrum of the product released by ß-elimination from the parasite mucin. The trans-sialylation reaction studied on the benzyl glycoside of 10 showed two monosialylated products whose relative abundance changed with time. On the other hand, only one product was produced by sialylation of the benzyl glycoside of 7. A preparative synthesis of the latter and spectroscopic analysis of the product unequivocally established the sialylation site at the less hindered (1→3)-linked galactopyranose.

Induction of IL-12 production in human peripheral monocytes by Trypanosoma cruzi Is mediated by glycosylphosphatidylinositol-anchored mucin-like glycoproteins and potentiated by IFN- γ and CD40-CD40L interactions.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), is characterized by immunopathology driven by IFN-γ secreting Th1-like T cells. T. cruzi has a thick coat of mucin-like glycoproteins covering its surface, which plays an important role in parasite invasion and host immunomodulation. It has been extensively described that T. cruzi or its products-like GPI anchors isolated from GPI-anchored mucins from the trypomastigote life cycle stage (tGPI-mucins)-are potent inducers of proinflammatory responses (i.e., cytokines and NO production) by IFN-γ primed murine macrophages. However, little is known about whether T. cruzi or GPI-mucins exert a similar action in human cells. We therefore decided to further investigate the in vitro cytokine production profile from human mononuclear cells from uninfected donors exposed to T. cruzi as well as tGPI-mucins. We observed that both living T. cruzi trypomastigotes and tGPI-mucins are potent inducers of IL-12 by human peripheral blood monocytes and this effect depends on CD40-CD40L interaction and IFN-γ. Our findings suggest that the polarized T1-type cytokine profile seen in T. cruzi infected patients might be a long-term effect of IL-12 production induced by lifelong exposure to T. cruzi tGPI-mucins.

Sugar-mediated disassembly of mucin/lectin multilayers and their use as pH-Tolerant, on-demand sacrificial layers.

The layer-by-layer (LbL) assembly of thin films on surfaces has proven to be an extremely useful technology for uses ranging from optics to biomedical applications. Releasing these films from the substrate to generate so-called free-standing multilayer films opens a new set of applications. Current approaches to generating such materials are limited because they can be cytotoxic, difficult to scale up, or have undesirable side reactions on the material. In this work, a new sacrificial thin film system capable of chemically triggered dissolution at physiological pH of 7.4 is described. The film was created through LbL assembly of bovine submaxillary mucin (BSM) and the lectin jacalin (JAC) for a (BSM/JAC) multilayer system, which remains stable over a wide pH range (pH 3-9) and at high ionic strength (up to 5 M NaCl). This stability allows for subsequent LbL assembly of additional films in a variety of conditions, which could be released from the substrate by incubation in the presence of a competitive inhibitor sugar, melibiose, which selectively disassembles the (BSM/JAC) section of the film. This novel multilayer system was then applied to generate free-standing, 7 µm diameter, circular ultrathin films, which can be attached to a cell surface as a "backpack". A critical thickness of about 100 nm for the (BSM/JAC) film was required to release the backpacks from the glass substrate, after incubation in melibiose solution at 37 °C for 1 h. Upon their release, backpacks were subsequently attached to murine monocytes without cytotoxicity, thereby demonstrating the compatibility of this mucin-based release system with living cells.

Role of lubricants on friction between self-ligating brackets and archwires.

OBJECTIVE: To evaluate the effect of different lubricants on friction between orthodontic brackets and archwires. MATERIALS AND METHODS: Active (Quick, Forestadent) and passive (Damon 3MX, Ormco) self-ligating brackets underwent friction tests in the presence of mucin- and carboxymethylcellulose (CMC)-based artificial saliva, distilled water, and whole human saliva (positive control). Dry friction (no lubricant) was used as the negative control. Bracket/wire samples (0.014 × 0.025 inch, CuNiTi, SDS Ormco) underwent friction tests eight times in a universal testing machine. RESULTS: Two-way analysis of variance showed no significant interaction between bracket type and lubricant (P  =  .324). Friction force obtained with passive self-ligating brackets was lower than that for active brackets (P < .001). Friction observed in the presence of artificial saliva did not differ from that generated under lubrication with natural human saliva, as shown by Tukey test. Higher friction forces were found with the use of distilled water or when the test was performed under dry condition (ie, with no lubricant). CONCLUSION: Lubrication plays a role in friction forces between self-ligating brackets and CuNiTi wires, with mucin- and CMC-based artificial saliva providing a reliable alternative to human natural saliva.

Mucin-like peptides from Echinococcus granulosus induce antitumor activity.

There is substantial evidence suggesting that certain parasites can have antitumor properties. We evaluated mucin peptides derived from the helminth Echinococcus granulosus (denominated Egmuc) as potential inducers of antitumor activity. We present data showing that Egmuc peptides were capable of inducing an increase of activated NK cells in the spleen of immunized mice, a fact that was correlated with the capacity of splenocytes to mediate killing of tumor cells. We demonstrated that Egmuc peptides enhance LPS-induced maturation of dendritic cells in vitro by increasing the production of IL-12p40p70 and IL-6 and that Egmuc-treated DCs may activate NK cells, as judged by an increased expression of CD69. This evidence may contribute to the design of tumor vaccines and open new horizons in the use of parasite-derived molecules in the fight against cancer.