Novel Animal Model of Limbal Stem Cell Deficiency Induced by Forcing Eye-Open at Birth.

PURPOSE: The aim of this study was to develop a rat model of limbal stem cell deficiency (LSCD) by forcing eye-open at birth (FEOB). METHODS: A total of 200 Sprague-Dawley neonatal rats were randomly divided into the control group and the experimental group, which received eyelid open surgery on postnatal day 1 (P1). Observation time points were defined as P1, P5, P10, P15, and P30. Slit-lamp microscope and corneal confocal microscope were used to observe the clinical features of the model. The eyeballs were collected for hematoxylin and eosin staining and periodic acid-Schiff staining. Proliferating cell nuclear antigen, CD68/polymorphonuclear leukocytes, and cytokeratin 10/12/13 immunostaining were performed, while the ultrastructure of the cornea was observed by scanning electron microscopy. Real-time polymerase chain reactions (PCRs), western blot, and immunohistochemical staining of activin A receptor-like kinase-1/5 were used to analyze the possible pathogenesis. RESULTS: FEOB could successfully induce the typical manifestations of LSCD, including corneal neovascularization, severe inflammation, and corneal opacity. In the FEOB group, goblet cells could be detected in the corneal epithelium by periodic acid-Schiff staining. The expression of cytokeratins was also different between the 2 groups. Furthermore, proliferating cell nuclear antigen immunohistochemical staining revealed the weak proliferation and differentiation ability of limbal epithelial stem cells in the FEOB group. Real-time PCRs, western blot, and immunohistochemical staining of activin A receptor-like kinase-1/activin A receptor-like kinase-5 in the FEOB group showed different expression patterns than those of the control group. CONCLUSIONS: FEOB in rats induces ocular surface changes resembling LSCD in humans, representing a novel model of LSCD.

Estrogen deficiency aggravates fluoride-induced small intestinal mucosa damage and junctional complexes proteins expression disorder in rats.

To investigate the effect of estrogen deficiency on the small intestinal mucosal barrier induced by fluoride (F), F exposure models of ovariectomy (OVX) rats (surgically removed ovaries) and non-OVX rats (normal condition) were established by adding sodium fluoride (NaF) (0, 25, 50, and 100 mg/L, calculated by F ion) in drinking water for 90 days. The intestinal mucosal histomorphology, mucosal barrier function, and protein expression levels of tight junctions (TJs), adhesion junctions (AJs), and desmosomes were evaluated in the duodenum, jejunum, and ileum. Hematoxylin-eosin (HE) staining and 5-Bromo-2-deoxyUridine (BrdU) measurement showed that excessive F-induced damage to intestinal epithelial cells and inhibited the proliferation of intestinal epithelial cells, eventually decreasing the number of goblet cells and decreasing glycoprotein secretion, as indicated by Alcian blue and periodic acid-Schiff (AB-PAS) and periodic acid-Schiff (PAS) staining. Further immunofluorescence analysis demonstrated that excessive F decreased the protein expression levels of occludin, zonula occludens-1 (ZO-1), E-cadherin, and desmoplakin (P < 0.05, P < 0.01) and enhanced the expression of claudin-2 (P < 0.01), suggesting that cell-to-cell junctions were disrupted. Collectively, F exposure impaired the small intestinal mucosal barrier by inducing damage to intestinal epithelial cells and inhibiting intestinal epithelial cell proliferation. Disorders in the junctional complex protein expression blocked the synergy between intercellular communication and aggravated mucosal injury. In particular, estrogen deficiency exacerbated F-induced enterotoxicity, which provides new explanations for the development and severity of intestinal disease in postmenopausal women with high-F areas.

Danshen injection induces autophagy in podocytes to alleviate nephrotic syndrome via the PI3K/AKT/mTOR pathway.

BACKGROUND: Danshen injection (DSI) is an agent extracted from the Salvia miltiorrhiza Bunge, a natural drug commonly used to alleviate kidney diseases. However, the material basis and therapeutic effects of DSI on nephrotic syndrome (NS) remain unclear. PURPOSE: To investigate the material basis of DSI and the therapeutic effects and underlying mechanisms of NS. METHODS: NS models were established using adriamycin-induced BALB/c mice and lipopolysaccharide-induced mouse podocytes (MPC-5). Following DSI and prednisone administration, kidney coefficients, 24 h urine protein, blood urea nitrogen, and serum creatinine levels were tested. Histomorphology was observed by periodic acid-Schiff staining and hematoxylin and eosin staining of the kidney sections. The glomerular basement membrane and autophagosomes of the kidneys were observed using transmission electron microscopy. Nephrin and desmin levels in the glomeruli were tested using immunohistochemistry. The viability of MPC-5 cells was tested using cell counting kit-8 after chloroquine and rapamycin administration in combination with DSI. The in vivo and in vitro protein levels of phosphatidylinositol 3-kinase (PI3K), AKT, phosphorylated AKT (Ser473), mammalian target of rapamycin (mTOR), microtubule-associated protein light chain 3 (LC3), beclin1, cleaved caspase-3, and caspase-3 were detected using western blotting. RESULTS: Our results showed that DSI contained nine main components: caffeic acid, danshensu, lithospermic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B, salvianolic acid C, salvianolic acid D, and 3, 4-Dihydroxybenzaldehyde. In in vivo studies, the NS mice showed renal function and pathological impairment. Podocytes were damaged, with decreased levels of autophagy and apoptosis, accompanied by inhibition of the PI3K/AKT/mTOR signaling. DSI administration resulted in improved renal function and pathology in NS mice, with the activation of autophagy and PI3K/AKT/mTOR signaling in the kidneys. Additionally, podocytes were less damaged and intracellular autophagosomes were markedly increased. In vitro studies have shown that DSI activated MPC-5 autophagy and reduced apoptosis via the PI3K/AKT/mTOR pathway. CONCLUSION: Collectively, this study demonstrated that DSI activated podocyte autophagy and reduced apoptosis via the PI3K/AKT/mTOR signaling, ultimately attenuating NS. Our study clarified the main components of DSI and elucidated its therapeutic effects and potential mechanisms for NS, providing new targets and agents for the clinical treatment of NS.

Pathological Comparison of Rat Pulmonary Models Induced by Silica Nanoparticles and Indium-Tin Oxide Nanoparticles.

Purpose: The objective of this study was to evaluate and compare the histopathological implications of silica nanoparticles (Nano-SiO2) and indium-tin oxide nanoparticles (Nano-ITO), in vivo. Methods: Male Sprague-Dawley rats were exposed to Nano-SiO2 (50 mg/kg) and Nano-ITO (6 mg/kg) by a single intratracheal instillation, respectively. Broncho-alveolar lavage fluid (BALF) and lung tissue were obtained at 7, 14, 28, and 56 days post exposure for analysis of BALF inflammatory factors, total protein, and for lung tissue pathology. Histopathological and ultrastructural change in lungs were investigated by hematoxylin and eosin, Masson's trichrome, sirius red staining, periodic acid Schiff stain, and transmission electron microscopy. The expression of SP-A, collagen type I and III in lung tissue was determined by immunohistochemistry and ELISA. Results: The rats in both models exhibited obvious collagen fibrosis and the severity of the lung injury increased with time after exposure to respective dosage increased. Several parameters of pulmonary inflammation and fibrosis significantly increased in both groups, which was reflected by increased LDH activity, total proteins, TNF-α, and IL-6 levels in BALF, and confirmed by histopathological examination. The results also showed that the two models exhibited different features. Exposure to Nano-ITO caused persistent chronic lung inflammation, illustrated by the infiltration of a large amount of enlarged and foamy macrophages and neutrophils into the lung parenchyma. In Nano-SiO2 exposed rat lung tissue, granulomatous inflammation was most prominent followed by progressive and massive fibrotic nodules. Compared with the Nano-SiO2 rats, Nano-ITO exposed rats exhibited significantly severe pulmonary alveolar proteinosis (PAP) pathological changes, lower fibrosis, and higher levels of inflammatory biomarkers. However, Nano-SiO2 exposed rats had greater fibrosis pathological changes and more severe granulomas than Nano-ITO exposed rats. Conclusion: This study suggests that the Nano-SiO2-induced model has greater value in research into granulomas and fibrosis, while the Nano-ITO-induced model has greater repeatability in area of PAP.

Oxysophocarpine inhibits airway inflammation and mucus hypersecretion through JNK/AP-1 pathway in vivo and in vitro.

Asthma is a high-incidence disease in the world. Oxysophocarpine (OSC), a quinolizidine alkaloid displays various pharmacological functions including anti-inflammation, neuroprotective, anti-virus and antioxidant. Here, we established mice and cell asthmatic model to explore the effects of OSC for asthma treatment. Mice were sensitized and challenged with ovalbumin (OVA) and treated with OSC before challenge. Enzyme-linked immuno sorbent assay (ELISA), hematoxylin and eosin (H&E), periodic acid-schiff (PAS), tolonium chloride staining and immunohistochemical assay were performed. OSC treatment inhibited inflammatory cell infiltration and mucus secretion in the airway, reduced IgE level in mouse serum and decreased IL-4, IL-5 production in bronchoalveolar lavage fluid (BALF). OSC also reduced the spleen index to regulate immune function. Meanwhile, NCI-H292 cells were induced by lipopolysaccharide (LPS) to simulate airway epithelial injury. OSC pretreatment decreased the IL-6 and IL-8 cytokine levels, mucin 5 AC expression, and mucin 5 AC mRNA level in the cell model. Further, OSC suppressed the phosphorylation of c-Jun N-terminal kinase (JNK), and activator protein 1 (AP-1, Fos and Jun). These findings revealed that OSC alleviated bronchial asthma associated with JNK/AP-1 signaling pathway.

Ticagrelor and Dapagliflozin Have Additive Effects in Ameliorating Diabetic Nephropathy in Mice with Type-2 Diabetes Mellitus.

PURPOSE: Ticagrelor and dapagliflozin can suppress the activation of the NOD-like receptor 3 (NLRP3)-inflammasome and activate AMP-activated protein kinase (AMPK). The anti-inflammatory effects of dapagliflozin has been shown to depend on AMPK activation. Dapagliflozin and ticagrelor have been shown to have additive effects on the progression of diabetic cardiomyopathy in BTBR ob/ob mice with type-2 diabetes. We assessed whether dapagliflozin and ticagrelor have additive effects on the activation of the NLRP3-inflammasome and the progression of diabetic nephropathy in mice with type-2 diabetes. METHODS: Eight-week-old BTBR received either no-drug, dapagliflozin (1.5 mg/kg/d), ticagrelor (100 mg/kg/d), or their combination for 12 weeks. Blood was assessed weekly for glucose and urine for glucose and albumin. After 12 weeks, blood creatinine, cystatin C, inflammasome activation, and insulin were assessed by ELISA. Renal cortex samples were assessed by hematoxylin and eosin and periodic acid-Schiff staining. RT-PCR and immunoblotting were used to evaluate fibrosis and the activation of Akt, AMPK and the inflammasome. RESULTS: Both ticagrelor and dapagliflozin reduced serum creatinine and cystatin C levels and urinary albumin. Both drugs attenuated the increase in glomerular area and mesangial matrix index. Both drugs decreased collagen-1 and collagen-3 expression and the activation of the NLRP3-inflammasome. Both drugs increased P-AMPK levels, but only dapagliflozin increased P-Akt levels. Overall, the protective effects of dapagliflozin and ticagrelor were additive. CONCLUSIONS: Dapagliflozin and ticagrelor attenuated the progression of diabetic nephropathy in BTBR ob/ob mice with additive effects of the combination. This was associated with AMPK activation and reduced activation of the NLRP3 inflammasome, whereas only dapagliflozin increased Akt activation.

Structural characterisation of high affinity Siglec-2 (CD22) ligands in complex with whole Burkitt's lymphoma (BL) Daudi cells by NMR spectroscopy.

Siglec-2 undergoes constitutive endocytosis and is a drug target for autoimmune diseases and B cell-derived malignancies, including hairy cell leukaemia, marginal zone lymphoma, chronic lymphocytic leukaemia and non-Hodgkin's lymphoma (NHL). An alternative to current antibody-based therapies is the use of liposomal nanoparticles loaded with cytotoxic drugs and decorated with Siglec-2 ligands. We have recently designed the first Siglec-2 ligands (9-biphenylcarboxamido-4-meta-nitrophenyl-carboxamido-Neu5Acα2Me, 9-BPC-4-mNPC-Neu5Acα2Me) with simultaneous modifications at C-4 and C-9 position. In the current study we have used Saturation Transfer Difference (STD) NMR spectroscopy to monitor the binding of 9-BPC-4-mNPC-Neu5Acα2Me to Siglec-2 present on intact Burkitt's lymphoma Daudi cells. Pre-treatment of cells with periodate resulted in significantly higher STD NMR signal intensities for 9-BPC-4-mNPC-Neu5Acα2Me as the cells were more susceptible to ligand binding because cis-binding on the cell surface was removed. Quantification of STD NMR effects led to a cell-derived binding epitope of 9-BPC-4-mNPC-Neu5Acα2Me that facilitated the design and synthesis of C-2, C-3, C-4 and C-9 tetra-substituted Siglec-2 ligands showing an 88-fold higher affinity compared to 9-BPC-Neu5Acα2Me. This is the first time a NMR-based binding study of high affinity Siglec-2 (CD22) ligands in complex with whole Burkitt's lymphoma Daudi cells has been described that might open new avenues in developing tailored therapeutics and personalised medicine.

PstS-1, the 38-kDa Mycobacterium tuberculosis glycoprotein, is an adhesin, which binds the macrophage mannose receptor and promotes phagocytosis.

Mycobacterium tuberculosis, the primary causative agent of tuberculosis, infects macrophages and transforms the hostile intracellular environment into a permissive niche. M. tuberculosis infects macrophages using a variety of microbial ligand/cell receptor systems. In this study, binding assays with biotin-labelled mycobacterial cell wall proteins revealed five Concanavalin A-reactive proteins that bind macrophages. Among these proteins, we identified PstS-1, a 38-kDa M. tuberculosis mannosylated glycolipoprotein, and characterized it as an adhesin. Inhibition assays with mannan and immunoprecipitation demonstrated that PstS-1 binds the mannose receptor. We purified PstS-1 to 95.9% purity using ion exchange chromatography. The presence of mannose in purified PstS-1 was demonstrated by Concanavalin A interaction, which was abolished in the presence of sodium m-periodate and α-D-mannosidase. Gas chromatography revealed that purified PstS-1 contained 1% of carbohydrates by weight, which was mainly mannose. Finally, we used fluorescent microbeads coated with purified PstS-1 in phagocytosis assays and discovered that microbead uptake was inhibited by the pre-incubation of cells with GlcNAc, mannan and α-methyl mannoside. The interaction of PstS-1 coated beads with the mannose receptor was confirmed by confocal colocalization studies that showed high Pearson and Manders's colocalization coefficients. Our findings contribute to a better understanding of the strategies M. tuberculosis uses to infect host cells, the critical first step in the pathogenesis of tuberculosis.

The glycosylation pattern of common allergens: the recognition and uptake of Der p 1 by epithelial and dendritic cells is carbohydrate dependent.

Allergens are initiators of both innate and adaptive immune responses. They are recognised at the site of entry by epithelial and dendritic cells (DCs), both of which activate innate inflammatory circuits that can collectively induce Th2 immune responses. In an attempt to have a better understanding of the role of carbohydrates in the recognition and uptake of allergens by the innate immune system, we defined common glycosylation patterns in major allergens. This was done using labelled lectins and showed that allergens like Der p 1 (Dermatophagoides pteronyssinus group 1), Fel d 1 (Felis domisticus), Ara h 1 (Arachis hypogaea), Der p 2 (Dermatophagoides pteronyssinus group 2), Bla g 2 (Blattella germanica) and Can f 1 (Canis familiaris) are glycosylated and that the main dominant sugars on these allergens are 1-2, 1-3 and 1-6 mannose. These observations are in line with recent reports implicating the mannose receptor (MR) in allergen recognition and uptake by DCs and suggesting a major link between glycosylation and allergen recognition. We then looked at TSLP (Thymic Stromal Lymphopoietin) cytokine secretion by lung epithelia upon encountering natural Der p 1 allergen. TSLP is suggested to drive DC maturation in support of allergic hypersensitivity reactions. Our data showed an increase in TSLP secretion by lung epithelia upon stimulation with natural Der p 1 which was carbohydrate dependent. The deglycosylated preparation of Der p 1 exhibited minimal uptake by DCs compared to the natural and hyperglycosylated recombinant counterparts, with the latter being taken up more readily than the other preparations. Collectively, our data indicate that carbohydrate moieties on allergens play a vital role in their recognition by innate immune cells, implicating them in downstream deleterious Th2 cell activation and IgE production.

Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells.

5-hydroxymethylcytosine (5hmC) is a modified base present at low levels in diverse cell types in mammals. 5hmC is generated by the TET family of Fe(II) and 2-oxoglutarate-dependent enzymes through oxidation of 5-methylcytosine (5mC). 5hmC and TET proteins have been implicated in stem cell biology and cancer, but information on the genome-wide distribution of 5hmC is limited. Here we describe two novel and specific approaches to profile the genomic localization of 5hmC. The first approach, termed GLIB (glucosylation, periodate oxidation, biotinylation) uses a combination of enzymatic and chemical steps to isolate DNA fragments containing as few as a single 5hmC. The second approach involves conversion of 5hmC to cytosine 5-methylenesulphonate (CMS) by treatment of genomic DNA with sodium bisulphite, followed by immunoprecipitation of CMS-containing DNA with a specific antiserum to CMS. High-throughput sequencing of 5hmC-containing DNA from mouse embryonic stem (ES) cells showed strong enrichment within exons and near transcriptional start sites. 5hmC was especially enriched at the start sites of genes whose promoters bear dual histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 4 trimethylation (H3K4me3) marks. Our results indicate that 5hmC has a probable role in transcriptional regulation, and suggest a model in which 5hmC contributes to the 'poised' chromatin signature found at developmentally-regulated genes in ES cells.

Improvement of the CuZn-superoxide dismutase enzyme activity and stability as a therapeutic agent by modification with polysialic acids.

The optimal process for the polysialylation reaction was as follows: polysialicacid (PSA) was activated by periodate oxidation, then coupled to CuZn superoxide dismutase (SOD) with a PSA:SOD molar ratio of 40:1 for 24 h. The resulting polysialylated protein contained 3.9 ± 0.3 mol PSA per mol SOD. SDS-PAGE and atomic force microscopy revealed that the molecular weight of polysialylated SOD was about 90-100 kDa. The average size was 10-15 nm, about four-fold of the native enzyme. Compared to the native enzyme, the activity and stability of the polysialylated SOD, as well as resistance to heat, acid, alkali and proteases present in human digestive system such as pepsin and trypsin, were improved significantly as therapeutic agent.

Purified chicken intestinal mucin attenuates Campylobacter jejuni pathogenicity in vitro.

Campylobacter jejuni is a major causative agent of diarrhoeal disease worldwide in the human population. In contrast, heavy colonization of poultry typically does not lead to disease and colonized chickens are a major source of Campylobacter infections in humans. Previously, we have shown that chicken (but not human) intestinal mucus inhibits C. jejuni internalization. In this study, we test the hypothesis that chicken mucin, the main component of mucus, is responsible for this inhibition of C. jejuni virulence. Purified chicken intestinal mucin attenuated C. jejuni binding and internalization into HCT-8 cells depending on the site of origin of the mucin (large intestine>small intestine>caecum). C. jejuni invasion of HCT-8 cells was preferentially inhibited compared to bacterial binding to cells. Exposure of the mucin to sodium metaperiodate recovered bacterial invasion levels, suggesting a glycan-mediated effect. However, fucosidase or sialidase pre-treatment of mucin failed to abrogate the inhibition of C. jejuni pathogenicity. In conclusion, differences in the composition of chicken and human intestinal mucin may contribute to the differential outcome of Campylobacter infection of these hosts.

Structural stability of Sclerotium rolfsii ATCC 201126 beta-glucan with fermentation time: a chemical, infrared spectroscopic and enzymatic approach.

AIMS: Sclerotium rolfsii ATCC 201126 exopolysaccharides (EPSs) recovered at 48 h (EPS I) and 72 h (EPS II) of fermentation, with differences in rheological parameters, hydrogel topography, salt tolerance, antisyneresis, emulsifying and suspending properties, were subjected to a polyphasic characterization in order to detect structural divergences. METHODS AND RESULTS: Fermenter-scale production led to productivity (P(r)) and yield (Y(P/C)) values higher at 48 h (P(r) = 0.542 g l(-1) h(-1); Y(P/C) = 0.74) than at 72 h (P(r) = 0.336 g l(-1) h(-1); Y(P/C) = 0.50). Both EPSs were neutral glucose-homopolysaccharides with a beta-(1,3)-glycosidic backbone and single beta-(1,6)-glucopyranosyl sidechains regularly attached every three residues in the main chain, as revealed by chemical analyses. The infra-red diagnostic peak at 890 cm(-1) confirmed beta-glycosidic linkages, while gentiobiose released by beta-(1,3)-glucanases confirmed single beta-1,6-glycosidic branching for both EPSs. CONCLUSIONS: The true modular repeating unit of S. rolfsii ATCC 201126 scleroglucan could be resolved. Structural stability was corroborated and no structural differences could be detected as to account for the variations in EPSs behaviour. SIGNIFICANCE AND IMPACT OF THE STUDY: Recovery of S. rolfsii ATCC 201126 scleroglucan at 48 h might be considered based on better fermentation kinetic parameters and no detrimental effects on EPS structural features.

Kinetic characterization of the oxidation of chlorogenic acid by polyphenol oxidase and peroxidase. Characteristics of the o-quinone.

Chlorogenic acid is the major diphenol of many fruits, where it is oxidized enzymatically by polyphenol oxidase (PPO) or peroxidase (POD) to its o-quinone. In spectrophotometric studies of chlorogenic acid oxidation with a periodate ratio of [CGA]0/[IO4-]0 < 1 and [CGA]0/[IO4-]0 > 1, the o-quinone was characterized as follows: lambda(max) at 400 nm and epsilon = 2000 and 2200 M-1 cm-1 at pH 4.5 and 7.0, respectively. In studies of o-quinone generated by the oxidation of chlorogenic acid using a periodate at ratio of [CGA]0/[IO4-]0 > 1, a reaction with the remaining substrate was detected, showing rate constants of k = 2.73 +/- 0.17 M-1 s-1 and k' = 0.05 +/- 0.01 M-1 s-1 at the above pH values. A chronometric spectrophotometric method is proposed to kinetically characterize the action of the PPO or POD on the basis of measuring the time it takes for a given amount of ascorbic acid to be consumed in the reaction with the o-quinone. The kinetic constants of mushroom PPO and horseradish POD are determined.

Kinetic characterization of the oxidation of esculetin by polyphenol oxidase and peroxidase.

Esculetin has been described as an inhibitor of tyrosinase and polyphenol oxidase and, therefore, of melanogenesis. In this work, we demonstrate that esculetin is not an inhibitor but a substrate of mushroom polyphenol oxidase (PPO) and horseradish peroxidase (POD), enzymes which oxidize esculetin, generating its o-quinone. Since o-quinones are very unstable, the usual way of determining the enzymatic activity (slope of recordings) is difficult. For this reason, we developed a chronometric method to characterize the kinetics of this substrate, based on measurements of the lag period in the presence of micromolar concentrations of ascorbic acid. The catalytic constant determined was of the same order for both enzymes. However, polyphenol oxidase showed greater affinity (a lower Michaelis constant) than peroxidase for esculetin. The affinity of PPO and POD towards oxygen and hydrogen peroxide was very high, suggesting the possible catalysis of both enzymes in the presence of low physiological concentrations of these oxidizing substrates. Taking into consideration optimum pHs of 4.5 and 7 for POD and PPO respectively, and the acidic pHs of melanosomes, the studies were carried out at pH 4.5 and 7. The in vivo pH might be responsible for the stronger effect of these enzymes on L-tyrosine and L-3,4-dihydroxyphenylanaline (L-DOPA) (towards melanogenesis) and on cumarins such as esculetin towards an alternative oxidative pathway.

Susceptibility of staphylococcal biofilms to enzymatic treatments depends on their chemical composition.

Bacterial infections are serious complications after orthopaedic implant surgery. Staphylococci, with Staphylococcus epidermidis as a leading species, are the prevalent and most important species involved in orthopaedic implant-related infections. The biofilm mode of growth of these bacteria on an implant surface protects the organisms from the host's immune system and from antibiotic therapy. Therapeutic agents that disintegrate the biofilm matrix would release planktonic cells into the environment and therefore allow antibiotics to eliminate the bacteria. An addition of a biofilm-degrading agent to a solution used for washing-draining procedures of infected orthopaedic implants would greatly improve the efficiency of the procedure and thus help to avoid the removal of the implant. We have previously shown that the extracellular staphylococcal matrix consists of a poly-N-acetylglucosamine (PNAG), extracellular teichoic acids (TAs) and protein components. In this study, we accessed the sensitivity of pre-formed biofilms of five clinical staphylococcal strains associated with orthopaedic prosthesis infections and with known compositions of the biofilm matrix to periodate, Pectinex Ultra SP, proteinase K, trypsin, pancreatin and dispersin B, an enzyme with a PNAG-hydrolysing activity. We also tested the effect of these agents on the purified carbohydrate components of staphylococcal biofilms, PNAG and TA. We found that the enzymatic detachment of staphylococcal biofilms depends on the nature of their constituents and varies between the clinical isolates. We suggest that a treatment with dispersin B followed by a protease (proteinase K or trypsin) could be capable to eradicate biofilms of a variety of staphylococcal strains on inert surfaces.

Extracellular ATP has stimulatory effects on the expression and release of IL-6 via purinergic receptors in normal human epidermal keratinocytes.

Extracellular ATP regulates proliferation and differentiation, functioning as an important messenger via purinergic (P2) receptors in keratinocytes. In this study, we investigated the effects of ATP on cytokine production in cultured normal human epidermal keratinocytes (NHEKs). Adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), adenosine 5'-O-2-(thio)diphosphate (ADPbetaS), ADP, ATP, and 2', 3'-O-(4-benzoyl-benzoyl) ATP (BzATP) significantly increased the release of IL-6. The P2 antagonists, suramin-, reactive blue 2-, and periodate-oxidized ATP, inhibited ATP-induced IL-6 release, whereas pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, adenosine 3'-phosphate 5'-phosphate, 1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine, and pertussis toxin did not. SQ22563, an adenylate cyclase inhibitor, inhibited ATP-induced IL-6 release. ATPgammaS, ADPbetaS, ATP, and BzATP significantly increased the intracellular cAMP content. Reverse transcription-PCR showed expression of P2Y1, P2Y2, P2Y4, P2Y11, P2Y12, P2Y13, P2X1, P2X4, P2X5, P2X6, and P2X7 receptor subtypes. Additionally, UVB radiation evoked the release of ATP from NHEKs. The release of IL-6 and the expression of IL-6 mRNA were increased after UVB radiation, and these increases were also inhibited by P2 receptor antagonists. These results suggest that cAMP-generating P2Y receptors are likely functional in ATP-induced IL-6 production in NHEKs. Furthermore, in UVB-radiated cells, we note the possibility that P2 receptor antagonists may reduce skin inflammation.

Echinococcus granulosus coproantigens: chromatographic fractionation and characterization.

Dogs infected with adult tapeworms of Echinococcus granulosus release antigens (coproantigens) in faeces which can be detected by a capture ELISA. Supernatants prepared from E. granulosus-infected dog faecal samples were fractionated by size-exclusion fast protein liquid chromatography (FPLC) on a Superose-6 column. Coproantigen ELISA and Western blotting were used to demonstrate the immunoreactivity of eluted fractions. Two main FPLC peaks of antigenic activity were detected and designated as fraction F1 and fraction F2 with approximate relative molecular weights > 670 kDa, and in the range of 146 to 440 kDa respectively. These two antigenic fractions (F1 and F2) fractionated from infected dog faeces were heat stable and largely protease-insensitive, but were highly sensitive to sodium periodate treatment, which strongly suggested the involvement of carbohydrates. Capture IgG antibodies against E. granulosus proglottis somatic extracts, detected a molecule with an approximate molecular weight of 155 kDa in fraction F2 after immunoblotting. The 155 kDa antigen could be completely ablated by sodium periodate treatment, but not after protease or lipase treatment. A surface tegument preparation of adult E. granulosus tapeworms contained large amounts of antigen that corresponded in size range and antigenicity to that observed in the FPLC fraction F2. There was also a peak of antigenic activity at > 670 kDa corresponding to fraction F1 from a culture derived excretory-secretory (E-S) adult tapeworm preparation. The involvement of carbohydrate moieties in coproantigen activity present in the FPLC fractions F1 and F2 from faecal supernatants of E. granulosus-infected dogs was confirmed by lectin-binding assays and exoglycosidase treatment, which showed that alpha-D-mannose and/or alpha-D-glucose, beta-galactose and N-acetyl-beta-glucosamine residues were the most important carbohydrate components in putative coproantigens present in both fractions. N-acetyl-beta-glucosamine and sialic acid residues were also contained in coproantigen molecules present in fraction F2. These results suggested that coproantigens detected in faeces of E. granulosus-infected dogs are large molecular weight molecules that may be derived from the carbohydrate-rich surface glycocalyx of adult worms, and are shed, released or secreted during the life-span of the tapeworm.

Adherence of Brucella to human epithelial cells and macrophages is mediated by sialic acid residues.

The basis for the interaction of Brucella species with the surface of epithelial cells before migration in the host within polymorphonuclear leucocytes is largely unknown. Here, we studied the ability of Brucella abortus and Brucella melitensis to adhere to cultured epithelial (HeLa and HEp-2) cells and THP-1-derived macrophages, and to bind extracellular matrix proteins (ECM). The brucellae adhered to epithelial cells forming localized bacterial microcolonies on the cell surface, and this process was inhibited significantly by pretreatment of epithelial cells with neuraminidase and sodium periodate and by preincubation of the bacteria with heparan sulphate and N-acetylneuraminic acid. Trypsinization of epithelial cells yielded increased adherence, suggesting unmasking of target sites on host cells. Notably, the brucellae also adhered to cultured THP-1 cells, and this event was greatly reduced upon removal of sialic acid residues from these cells with neuraminidase. B. abortus bound in a dose-dependent manner to immobilized fibronectin and vitronectin and, to a lesser extent, to chondroitin sulphate, collagen and laminin. In sum, our data strongly suggest that the adherence mechanism of brucellae to epithelial cells and macrophages is mediated by cellular receptors containing sialic acid and sulphated residues. The recognition of ECM (fibronectin and vitronectin) by the brucellae may represent a mechanism for spread within the host tissues. These are novel findings that offer new insights into understanding the interplay between Brucella and host cells.

Syndecan-1/CD138 expression in normal myeloid, acute lymphoblastic and myeloblastic leukemia cells.

Stabilization of cell surface antigens and preservation of ultrastructural integrity are important aspects of immunoelectron microscopical studies. In the present study, 4 anti-syndecan-1/CD138 (B-B2, B-B4, MI15, 1D4) monoclonal antibodies (mAbs) were applied in combination with periodatelysine-paraformaldehyde (PLP) fixation and indirect pre-embedding peroxidase electron microscopical immunocytochemistry to analyse the localization and function of these molecules in normal myeloid cells, acute lymphoblastic leukemia (ALL) cells and acute myeloblastic leukemia (AML) cells. One case of normal human bone marrow, 3 cases of untreated AML and 2 cases of untreated ALL were studied. Samples were immediately fixed for 4 h in freshly-prepared PLP fixative in 0.037 mol/L phosphate buffer, pH 7.4, containing 10 mmol/L sodium metaperiodate, 75 mmol/L lysine, and 2% paraformaldehyde. Expression of syndecan-1 was found at the plasma membrane of all cell types. Staining intensity at the membrane of AML cells was stronger than that on the membrane of normal myeloid and ALL cells. We conclude that anti-syndecan-1/CD138 mAbs in combination with the method described here are a suitable tool for detection of cell surface syndecan molecules in cells originating from progenitor cells that can differentiate in both myeloid and lymphoid cells.