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ANALISI DEI GLICOSAMINOGLICANI DI FLUIDI E ORGANI BIOLOGICI IN UN MODELLO ANIMALE DI MUCOPOLISACCARIDOSI II (SINDROME DI HUNTER): EFFETTO DEI TRATTAMENTI CON TERAPIA ENZIMA SOSTITUTIVA E GENISTEINA
No full textLa mucopolisaccaridosi II è una malattia genetica recessiva legata al cromosoma X, causata dal deficit dell'enzima lisosomiale iduronato-2-solfatasi (I2S). La mancanza di I2S fa sì che i glicosaminoglicani (GAG) come il condroitin solfato (CS), il dermatan solfato (DS) e l'eparan solfato (HS) si accumulino in tutti i tessuti del corpo, causando anomalie a diversi organi. Per MPS II, la terapia enzimatica sostitutiva (ERT) sembra essere il trattamento più efficace. Tuttavia, ERT non è in grado di attraversare la barriera emato-encefalica e mostra diversi limiti. L'isoflavone della soia genisteina è stata studiato come potenziale terapia per le MPS grazie alla sua presunta capacità di inibire la sintesi dei GAG e il loro accumulo. Inoltre, Genisteina può attraversare la barriera ematoencefalica nei modelli murini, ma studi precedenti hanno mostrato esiti contrastanti.
Questo progetto è focalizzato sulla caratterizzazione qualitativa e quantitativa dei GAG in un modello murino di MPS II (I2S knock-out) non trattato, trattato con ERT o trattato con Genisteina.
I GAG sono stati estratti da campioni di plasma, urina, fegato e cervello di 225 topi, secondo un protocollo standardizzato. La frazione di GAG grezza è stata digerita con enzimi specifici per isolare le unità disaccaridiche di acido ialuronico (HA), CS, DS e HS. I disaccaridi sono stati poi liofilizzati e contrassegnati con un fluoroforo. Infine, i disaccaridi derivatizzati sono stati quantificati mediante elettroforesi capillare interfacciata con LIF (laser-induced fluorescence).
Rispetto al Wild Type (WT), i campioni I2S KO hanno mostrato sin dalle prime settimane differenze significative per tutti i parametri analizzati. In questi soggetti, la concentrazione di CS-DS, HS e HA, così come la concentrazione totale di GAG, è aumentata in modo significativo rispetto al WT e rimane elevata durante tutte le settimane analizzate. Si evidenzia una elevata concentrazione di GAG, uno squilibrio del rapporto CS / HS verso HS, un aumento della densità di carica e un’alterazione del profilo di solfatazione. In particolare, CS-DS è principalmente solfatato nella posizione 2,4s, rispetto alla posizione 6s che è prevalente nei soggetti WT. Queste differenze sono particolarmente pronunciate nei campioni di fegato, suggerendo un maggiore accumulo di GAG negli organi solidi. Per quanto riguarda le terapie, possiamo notare come l'ERT possa ridurre molti dei parametri alterati, ma non può ripristinare completamente la condizione di WT. Inoltre, ERT non ha determinato alterazioni nei campioni di cervello, confermando che l'enzima non può attraversare la barriera ematoencefalica. Il trattamento con Genisteina non ha mostrato alterazioni significative rispetto ai soggetti I2S KO non trattati.
In conclusione, confrontando i risultati ottenuti da topi WT e topi I2S KO, osserviamo valori più elevati per ciascun indice considerato. Possiamo anche evidenziare come l'aumento dei disaccaridi solfatati in posizione 2,4s indichi un accumulo di DS in questi organi, specifico della MPSII. Da questi risultati si può proporre l'analisi dell'indice di solfatazione 2,4s nel plasma, come strumento utile nella diagnosi di MPSII. Inoltre, come nei pazienti con MPSII, la terapia ERT può ridurre significativamente l'accumulo di GAG. Questi risultati rafforzano ulteriormente la coerenza di questo modello animale con la patologia MPSII. Tuttavia, ERT non è in grado di indurre la normalizzazione totale dei parametri. Al contrario, la terapia con genisteina non ha mostrato differenze significative nei soggetti I2S KO, in nessuno dei parametri considerati. Nella fase successiva, analizzeremo diversi organi. Inoltre, avremo più indicazioni sull'efficacia di diverse terapie, fornendo le basi per futuri studi clinici sull'uomo.Mucopolysaccharidoses II is an X-linked recessive disorder caused by lysosomal enzyme iduronate-2-sulfatase (I2S) deficiency. The lack of I2S causes glycosaminoglycans (GAGs) such as chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulfate (HS) to accumulate in all body tissues, causing organ abnormalities. In severe cases, this leads to death during the teenage years. In MPS II, enzyme replacement therapy (ERT) appears to be the most effective treatment. However, ERT is unable to cross the blood-brain barrier and shows several limitations. The isoflavone genistein has been studied as a potential therapy for the MPS because of its putative ability to inhibit GAG synthesis and subsequent accumulation. Furthermore, genistein can cross the blood-brain barrier in murine models, but previous studies showed variable outcomes.
This project is focused on GAGs qualitative and quantitative characterization in a murine MPS II model (I2S knock-out) non-treated, treated with ERT or treated with genistein, analyzed at different weeks after the beginning of therapy.
GAGs were extracted from plasma, urine, liver and brain samples of 225 mice, according to a standardized protocol. The crude GAGs fraction was digested with specific enzymes to isolate Hyaluronic Acid (HA), CS, DS and HS disaccharide units. Disaccharides were lyophilized and tagged with a fluorophore. Finally, derivatized disaccharides were separated and quantified by capillary electrophoresis interfaced with laser-induced fluorescence.
Compared to wild type (WT), the I2S KO samples showed from the first weeks significant differences for all the analyzed parameters. In these subjects, CS-DS, HS and HA concentration, as well as total GAGs concentration, increased significantly compared to WT subjects and remains high during all the analyzed weeks. We observe, higher GAGs concentration, CS / HS imbalance towards HS, increase in charge density, and alteration in the sulfation profile. In particular, CS-DS was mainly sulfated in the 2,4s position, compared to the 6s position which was prevalent in WT subjects. These differences are particularly pronounced in liver samples, suggesting a higher GAGs accumulation in solid organs. As for the therapies, we can see how ERT can reduce many of the altered parameters, but it cannot restore the WT condition completely. Moreover, ERT did not show significant alterations in the brain samples, confirming that the enzyme cannot cross the blood-brain barrier. Genistein treatment, showed no significant alterations, either quantitatively or qualitatively, compared to non-treated I2S KO subjects.
Comparing the results obtained from WT mice and I2S KO mice we observe higher values of each index considered. We can also evidence how the increase in disaccharides sulfated in position 2,4s indicates an accumulation of DS in plasma, urine, liver and brain, specific of MPSII. From these results, we can propose the analysis of 2,4s sulfation index in plasma, as a useful tool in the diagnosis of this pathology. Moreover, as in MPSII patients, ERT therapy can significantly reduce GAGs accumulation. These findings further strengthen the coherence of this animal model to MPSII pathology. However, ERT is not able to induce total normalization of the parameters. On the contrary, genistein therapy did not show significant differences in I2S KO subjects, in any of the parameters considered. Therefore, this therapy would not seem to benefit the patient. In the next stage, we will also analyze different organs. Furthermore, we will have more indications of the effectiveness of different therapies in reducing GAGs accumulation, providing the basis for future clinical trials on humans
Chondroitin Sulfate and Glucosamine as Disease Modifying Anti- Osteoarthritis Dru gs (DMOADs).
No full textOsteoarthritis is a disabling affliction expected to increase in the coming decades, and disease- modifying osteoarthritis drugs (DMOADs) would be highly desirable adjuncts to symptomatic relief and structure reconstruction as they may delay the disease process. Chondroitin sulfate and glucosamine have been observed to exert beneficial effects on the metabolism of various cells involved in osteoarthritis as well as in animal models and clinical trials. Clinical trials have reported beneficial effects of both these biological agents, alone or in combination, on pain and functions as well as their structure-modifying capacity reported and analyzed in recent meta-analyses. Nonetheless, the effectiveness of these bioactive (macro)molecules as DMOADs reported from randomized trials is mismatched. Current studies with varying levels of evidence suggest that chondroitin sulfate and glucosamine can modify the disease progression but at the same time there are not absolute certainties on their efficacy in modifying the course of the disease. This comprehensive review aims to clarify the role of these compounds in the therapeutic molecules/ drugs useful to patients affected by osteoarthritis.Osteoarthritis is a disabling affliction expected to increase in the coming decades, and disease-modifying osteoarthritis drugs (DMOADs) would be highly desirable adjuncts to symptomatic relief and structure reconstruction as they may delay the disease process. Chondroitin sulfate and glucosamine have been observed to exert beneficial effects on the metabolism of various cells involved in osteoarthritis as well as in animal models and clinical trials. Clinical trials have reported beneficial effects of both these biological agents, alone or in combination, on pain and functions as well as their structure-modifying capacity reported and analyzed in recent meta-analyses. Nonetheless, the effectiveness of these bioactive (macro)molecules as DMOADs reported from randomized trials is mismatched. Current studies with varying levels of evidence suggest that chondroitin sulfate and glucosamine can modify the disease progression but at the same time there are not absolute certainties on their efficacy in modifying the course of the disease. This comprehensive review aims to clarify the role of these compounds in the therapeutic molecules/drugs useful to patients affected by osteoarthritis
Differences among Three Branded Formulations of Hyaluronic Acid: Data from Environmental Scanning Electron Microscope Profile, Rheology Behavior and Biological Activity
Full text linkBackground: This study has analysed the viscosupplemental proprieties of three
commercially available formulations of Hyaluronic Acid (HA) suspension (F1: Synvisc,
Hylan G-F 20; F2: Hyalgan; F3: Donegal HA 2.0), which differ in composition, Molecular
Weight (MW) and HA content.
Methods: Analyses were conducted using rheology measurements and Environmental
Scanning Electron Microscope (ESEM). The capacity of the three tested formulations to
inhibit specific Metalloproteases (MMPs) was also evaluated.
Results: F1 is the only sample showing viscoelastic properties but may have increased
immunogenicity attributable to the subsequent chemical cross-linking process that
enhances the MW. F2 and F3 show a lower viscosity compared to F1. F2 has the lowest
viscosity at low shear rate, the lower independence from the oscillatory stress and a
solution-like rheology behaviour. F3 display a solution behaviour. However, unlike F2, F3
crossover point falls in the middle of the frequency range of interest showing a considerable
rheological behaviour. The internal structure of F3 (pseudo-spongy thick filaments)
suggests that it has the ability to interact with a great water content. The crossover points
of the examined samples clearly reveal their different rheological behaviour, allowing their
classification in gel-like or solution-like materials. F3 has higher ability in inhibiting MMP-
2 and MMP-9 activity compared to F1 and F2, probably due to its specific MW and/or
higher HA concentration.
Conclusion: The three tested HA formulations differ in rheological properties and
inhibition of MMP-2 and MMP-9 activity. F3 seems to be the most appropriate formulation
for the treatment of osteoarthritis and rheumatoid arthritis
Analytical Methods for Assessing Chondroitin Sulfate in Human Plasma.
No full textChondroitin sulfate (CS) is a linear heteropolysaccharide of repeating disaccharide units bearing sulfate groups in various positions, commonly at C4 and/or C6 of galactosamine. CS plays important roles in various (patho)physiological processes also performing intriguing biological and therapeutical activities. Plasmatic CS is mainly composed of nonsulfated and 4-sulfated disaccharides. To obtain samples for the determination of CS amount and composition in blood/plasma, dried blood spot (DBS) could be used. DBSs have many advantages over other laboratory methods, allowing for large-scale population screening. Many analytical techniques may be used for the determination of CS. In particular, CE has proved to be a very attractive alternative separation technique for complex polysaccharide characterization. In this work, we compared CS levels between plasma and DBS samples, using CE equipped with the highly sensitive laser-induced fluorescence detector. CS from DBS differs from plasma CS owing to the high content of disaccharides sulfated in C4 and C6. This is due to the presence of the more sulfated CS derived from blood cellular fraction, in particular leukocytes. The identification and quantification of CS in blood plasma could be a useful prognostic and diagnostic tool in pathological conditions and for pharmacological applications
Recent advances on separation and characterization of human milk oligosaccharides.
No full textFree human milk oligosaccharides (HMOs) are unique due to their highly complex nature and important emerging biological and protective functions during early life such as prebiotic activity, pathogen deflection, and epithelial and immune cell modulation. Moreover, four genetically determined heterogeneous HMO secretory groups are known to be based on their structure and composition. Over the years, several analytical techniques have been applied to characterize and quantitate HMOs, including nuclear magnetic resonance spectroscopy, high-performance liquid chromatography (HPLC), high pH anion-exchange chromatography, off-line and on-line mass spectrometry (MS), and capillary electrophoresis (CE). Even if these techniques have proven to be efficient and simple, most glycans have no significant UV absorption and derivatization with fluorophore groups prior to separation usually results in higher sensitivity and an improved chromatographic/electrophoretic profile. Consequently, the analysis by HPLC/CE of derivatized milk oligosaccharides with different chromophoric active tags has been developed. However, UV or fluorescence detection does not provide specific structural information and this is a key point in particular related to the highly complex nature of the milk glycan mixtures. As a consequence, for a specific determination of complex mixtures of oligomers, analytical separation is usually required with evaluation by means of MS, which has been successfully applied to HMOs, resulting in efficient compositional analysis and profiling in various milk samples. This review aims to give an overview of the current state-of-the-art techniques used in HMO analysis
Recent advances in capillary electrophoresis separation of monosaccharides, oligosaccharides and polysaccharides
Full text linkThis article illustrates the basis and applications of methodologies for the analysis of simple and complex carbohydrates by means of CE. After a description of the most common and novel approaches useful for the analysis and characterization of carbohydrates, this review covers the recent advances in CE separation of monosaccharides, oligosaccharides, and polysaccharides. Various CE techniques are also illustrated for the study of carbohydrates derived from complex glyco-derivatives such as glycoproteins and glycolipids, essential for biopharmaceutical and glycoproteomics applications as well as for biomarker detection. Most glycans have no significant UV absorption, and derivatization with fluorophore groups prior to separation usually results in higher sensitivity and an improved electrophoretic profile. We also discuss the recent applications and separations by CE of derivatized simple and more complex carbohydrates with different chromophoric active tags. Overall, this review aims to give an overview of the most recent state-of-the-art techniques used in carbohydrate analysis by CE
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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