RESUMEN
Chondroitin sulfate is extracted from animal cartilaginous tissues and is commercialized as active principle against osteoarthritis. Its biological activity depends on its purity grade and could be altered by the presence of other glycosaminoglycans like keratan sulfate that could be contemporarily extracted from animal tissues or like hyaluronic acid that, instead, is added on purpose in food supplements. Although numerous methods are reported in literature for quality control analyses of chondroitin sulfate, few of them are able to detect other glycosaminoglycans. In this paper, for the first time, a new high-performance CE method was set up to quantify the chondroitin sulfate, the eventual keratan sulfate, and hyaluronic acid as intact chains: five chondroitin sulfate standards and 13 animal origin samples or food supplements from six different suppliers were analyzed. The new method was able to determine keratan sulfate similarly to a previously reported high-performance anion-exchange chromatography method, but in addition it showed the advantage to determine also the hyaluronic acid as never reported before.
Asunto(s)
Sulfatos de Condroitina/química , Suplementos Dietéticos/análisis , Electroforesis Capilar/métodos , Ácido Hialurónico/análisis , Sulfato de Queratano/análisis , Animales , Límite de Detección , Modelos Lineales , Reproducibilidad de los ResultadosRESUMEN
Retinitis pigmentosa (RP) is characterized by progressive loss of vision due to photoreceptor degeneration leading to secondary inflammation. The urokinase-type plasminogen activator (uPA) system contributes to retinal inflammation, but its role in RP is unknown. In the rd10 mouse model of RP, we addressed this question with the use of the peptide UPARANT designed to interact with the uPA system. UPARANT was systemically administered from post-natal day (PD) 10 to PD30 when its efficacy in RP rescue was investigated using electroretinographic recordings, Western blot and immunocytochemistry. Temporal profile of protein expression in the uPA system was also investigated. UPARANT reduced both Müller cell gliosis and up-regulated levels of inflammatory markers and exerted major anti-apoptotic effects without influencing the autophagy cascade. Rescue from retinal cell degeneration was accompanied by improved retinal function. No scotopic phototransduction was rescued in the UPARANT-treated animals as determined by the kinetic analysis of rod-mediated a-waves and confirmed by rod photoreceptor markers. In contrast, the cone photopic b-wave was recovered and its rescue was confirmed in the whole mounts using cone arrestin antibody. Investigation of the uPA system regulation over RP progression revealed extremely low levels of uPA and its receptor uPAR both of which were recovered by HIF-1α stabilization indicating that HIF-1 regulates the expression of the uPA/uPAR gene in the retina. Ameliorative effects of UPARANT were likely to occur through an inhibitory action on up-regulated activity of the αvß3 integrin/Rac1 pathway that was suggested as a novel target for the development of therapeutic approaches against RP.
Asunto(s)
Oligopéptidos/farmacología , Degeneración Retiniana/tratamiento farmacológico , Retinitis Pigmentosa/tratamiento farmacológico , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Inflamación/tratamiento farmacológico , Inflamación/genética , Inflamación/patología , Ratones , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Retina/efectos de los fármacos , Retina/patología , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Células Fotorreceptoras Retinianas Conos/patología , Degeneración Retiniana/genética , Degeneración Retiniana/patología , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/patología , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
The urokinase-type plasminogen activator (uPA) receptor (uPAR) participates to the mechanisms causing renal damage in response to hyperglycaemia. The main function of uPAR in podocytes (as well as soluble uPAR -(s)uPAR- from circulation) is to regulate podocyte function through αvß3 integrin/Rac-1. We addressed the question of whether blocking the uPAR pathway with the small peptide UPARANT, which inhibits uPAR binding to the formyl peptide receptors (FPRs) can improve kidney lesions in a rat model of streptozotocin (STZ)-induced diabetes. The concentration of systemically administered UPARANT was measured in the plasma, in kidney and liver extracts and UPARANT effects on dysregulated uPAR pathway, αvß3 integrin/Rac-1 activity, renal fibrosis and kidney morphology were determined. UPARANT was found to revert STZ-induced up-regulation of uPA levels and activity, while uPAR on podocytes and (s)uPAR were unaffected. In glomeruli, UPARANT inhibited FPR2 expression suggesting that the drug may act downstream uPAR, and recovered the increased activity of the αvß3 integrin/Rac-1 pathway indicating a major role of uPAR in regulating podocyte function. At the functional level, UPARANT was shown to ameliorate: (a) the standard renal parameters, (b) the vascular permeability, (c) the renal inflammation, (d) the renal fibrosis including dysregulated plasminogen-plasmin system, extracellular matrix accumulation and glomerular fibrotic areas and (e) morphological alterations of the glomerulus including diseased filtration barrier. These results provide the first demonstration that blocking the uPAR pathway can improve diabetic kidney lesion in the STZ model, thus suggesting the uPA/uPAR system as a promising target for the development of novel uPAR-targeting approaches.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Nefropatías Diabéticas/inducido químicamente , Nefropatías Diabéticas/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Estreptozocina/farmacología , Animales , Diabetes Mellitus Experimental/inducido químicamente , Inflamación/metabolismo , Riñón/metabolismo , Masculino , Podocitos/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/fisiología , Regulación hacia Arriba/fisiología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
The effectiveness of hyaluronic acid (HA), also called as hyaluronan, and its formulations on tissue regeneration and epidermal disease is well-documented. High-molecular-weight hyaluronan (HHA) is an efficient space filler that maintains hydration, serves as a substrate for proteoglycan assembly, and is involved in wound healing. Recently, an innovative hybrid cooperative complex (HCC) of high- and low-molecular-weight hyaluronan was developed that is effective in wound healing and bioremodeling. The HCC proposed here consisted of a new formulation and contained 1.6 ± 0.1 kDa HHA and 250 ± 7 kDa LHA (low molecular weight hyaluronic acid). We investigated the performance of this HCC in a novel in vitro HaCaT (immortalized human keratinocytes)/HDF (human dermal fibroblast) co-culture model to assess its ability to repair skin tissue lesions. Compared to linear HA samples, HCC reduced the biomarkers of inflammation (Transforming Growth Factor-ß (TGF-ß), Tumor Necrosis Factor receptor-α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8)), and accelerated the healing process. These data were confirmed by the modulation of metalloproteases (MMPs) and elastin, and were compatible with a prospectively reduced risk of scar formation. We also examined the expression of defensin-2, an antimicrobial peptide, in the presence of hyaluronan, showing a higher expression in the HCC-treated samples and suggesting a potential increase in antibacterial and immunomodulatory functions. Based on these in vitro data, the presence of HCC in creams or dressings would be expected to enhance the resolution of inflammation and accelerate the skin wound healing process.
Asunto(s)
Materiales Biocompatibles/química , Ácido Hialurónico/química , Sustancias Macromoleculares/química , Cicatrización de Heridas , Biomarcadores , Línea Celular , Técnicas de Cocultivo , Humanos , Hidrodinámica , Ensayo de Materiales , Reología , Análisis EspectralRESUMEN
BACKGROUND: Thermostable phosphotriesterase-like lactonases (PLLs) are able to degrade organophosphates and could be potentially employed as bioremediation tools and bioscavengers. But nowadays their manufacturing in high yields is still an issue that limits their industrial applications. In this work we aimed to set up a high yield production and purification biotechnological process of two recombinant PLLs expressed in E. coli, the wild type SacPox from Sulfolobus acidocaldarius and a triple mutated SsoPox C258L/I261F/W263A, originally from Sulfolobus solfataricus. To follow this aim new induction approaches were investigated to boost the enzyme production, high cell density fermentation strategies were set-up to reach higher and higher enzyme yields up to 22-L scale, a downstream train was studied to meet the requirements of an efficient industrial purification process. RESULTS: Physiological studies in shake flasks demonstrated that the use of galactose as inducer increased the enzyme concentrations up to 4.5 folds, compared to the production obtained by induction with IPTG. Optimising high cell density fed-batch strategies the production and the productivity of both enzymes were further enhanced of 26 folds, up to 2300 U·L- 1 and 47.1 U·L- 1·h- 1 for SacPox and to 8700 U·L- 1 and 180.6 U·L- 1·h- 1 for SsoPox C258L/I261F/W263A, and the fermentation processes resulted scalable from 2.5 to 22.0 L. After being produced and extracted from the cells, the enzymes were first purified by a thermo-precipitation step, whose conditions were optimised by response surface methodology. A following ultra-filtration process on 100 and 5 KDa cut-off membranes drove to a final pureness and a total recovery of both enzymes of 70.0 ± 2.0%, suitable for industrial applications. CONCLUSIONS: In this paper, for the first time, a high yield biotechnological manufacturing process of the recombinant enzymes SacPox and SsoPox C258L/I261F/W263A was set-up. The enzyme production was boosted by combining a new galactose induction approach with high cell density fed-batch fermentation strategies. An efficient enzyme purification protocol was designed coupling a thermo-precipitation step with a following membrane-based ultra-filtration process.
Asunto(s)
Hidrolasas de Triéster Fosfórico/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Sulfolobus acidocaldarius/enzimología , Sulfolobus solfataricus/enzimología , Proteínas Arqueales/genética , Proteínas Arqueales/aislamiento & purificación , Proteínas Arqueales/metabolismo , Técnicas de Cultivo Celular por Lotes/instrumentación , Técnicas de Cultivo Celular por Lotes/métodos , Biodegradación Ambiental , Precipitación Química , Cromatografía en Gel/métodos , Estabilidad de Enzimas , Escherichia coli/genética , Fermentación , Hidrolasas de Triéster Fosfórico/genética , Hidrolasas de Triéster Fosfórico/aislamiento & purificación , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfolobus acidocaldarius/genética , Sulfolobus solfataricus/genética , Ultrafiltración/métodosRESUMEN
BACKGROUND: Steatosis is a chronic liver disease that depends on the accumulation of intracellular fatty acids. Currently, no drug treatment has been licensed for steatosis; thus, only nutritional guidelines are indicated to reduce its progression. The aim of this study is to combine different nutraceutical compounds in order to evaluate their synergistic effects on a steatosis in vitro model compared to their separate use. In particular, three different formulations based on silymarin, curcumin, vitamin E, docosahexaenoic acid (DHA), choline, and phosphatidylcholine were assayed. METHODS: Human hepatocellular carcinoma cells (HepG2 cell line) were treated with a mixture of fatty acids in order to induce an in vitro model of steatosic cells, and then the amount of intracellular fat was evaluated by Oil Red O staining. The peroxisome proliferator-activated receptors α and γ (PPARα and γ) expression, closely correlated to lipid metabolism, was evaluated. The efficiency of these receptors was evaluated through the study of LPL mRNA expression, a marker involved in the lipolysis mechanism. Superoxide dismutase (SOD-2) and malondialdehydes (MDA) in lipid peroxidation were assayed as specific biomarkers of oxidative stress. In addition, experiments were performed using human liver cells stressed to obtain a steatosis model. In particular, the content of the intracellular fat was assayed using Oil Red O staining, the activation of PPARα and γ was evaluated through western blotting analyses, and the LPL mRNA expression level was analyzed through qRT-PCR. RESULTS: All formulations proved effective on lipid content reduction of about 35%. The oxidative stress damage was reduced by all the substances separately and even more efficiently by the same in formulation (i.e. Formulation 1 and Formulation 3, which reduced the SOD-2 expression and induced the PPARs activation). Lipid peroxidation, was reduced about 2 fold by foormulation2 and up to 5 fold by the others compared to the cells pretreated with H2O2.Formulation 1, was more effective on PPARγ expression (2.5 fold increase) respect to the other compounds on FA treated hepathocytes. Beside, LPL was activated also by Formulation 3 and resulted in a 5 to 9 fold-increase respect to FA treated control. CONCLUSIONS: Our results proved that the formulations tested could be considered suitable support to face steatosis disease beside the mandatory dietetic regimen.
Asunto(s)
Suplementos Dietéticos , Sinergismo Farmacológico , Hígado Graso/dietoterapia , Hígado Graso/tratamiento farmacológico , Colina/administración & dosificación , Curcumina/administración & dosificación , Ácidos Docosahexaenoicos/administración & dosificación , Combinación de Medicamentos , Composición de Medicamentos , Hígado Graso/metabolismo , Hígado Graso/patología , Células Hep G2 , Humanos , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/patología , PPAR alfa/genética , PPAR gamma/genética , Fosfatidilcolinas/administración & dosificación , Silimarina/administración & dosificación , Vitamina E/administración & dosificaciónRESUMEN
AIMS/HYPOTHESIS: Angiogenesis and inflammation characterise proliferative diabetic retinopathy (PDR), a major complication of diabetes mellitus. However, the impact of inflammation on the pathogenesis of PDR neovascularisation has not been elucidated. Here, we assessed the capacity of PDR vitreous fluid to induce pro-angiogenic/proinflammatory responses in endothelium and the contribution of the inflammation-related pattern recognition N-formyl peptide receptors (FPRs) in mediating these responses. METHODS: Pooled and individual pars plana vitrectomy-derived PDR vitreous fluid ('PDR vitreous') samples were assessed in endothelial cell proliferation, motility, sprouting and morphogenesis assays, and for the capacity to induce proinflammatory transcription factor activation, reactive oxygen species production, intercellular junction disruption and leucocyte-adhesion molecule upregulation in these cells. In vivo, the pro-angiogenic/proinflammatory activity of PDR vitreous was tested in murine Matrigel plug and chick embryo chorioallantoic membrane (CAM) assays. Finally, the FPR inhibitors Boc-Phe-Leu-Phe-Leu-Phe (Boc-FLFLF) and Ac-L-Arg-Aib-L-Arg-L-Cα(Me)Phe-NH2 tetrapeptide (UPARANT) were evaluated for their capacity to affect the biological responses elicited by PDR vitreous. RESULTS: PDR vitreous activates a pro-angiogenic/proinflammatory phenotype in endothelial cells. Accordingly, PDR vitreous triggers a potent angiogenic/inflammatory response in vivo. Notably, the different capacity of individual PDR vitreous samples to induce neovessel formation in the CAM correlates with their ability to recruit infiltrating CD45+ cells. Finally, the FPR inhibitor Boc-FLFLF and the novel FPR antagonist UPARANT inhibit neovessel formation and inflammatory responses triggered by PDR vitreous in the CAM assay. CONCLUSIONS/INTERPRETATION: This study provides evidence that inflammation mediates the angiogenic activity of PDR vitreous and paves the way for the development of FPR-targeting anti-inflammatory/anti-angiogenic approaches for PDR therapy.
Asunto(s)
Retinopatía Diabética/metabolismo , Inflamación/metabolismo , Receptores de Formil Péptido/metabolismo , Cuerpo Vítreo/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Línea Celular , Retinopatía Diabética/inmunología , Femenino , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Masculino , Ratones , Persona de Mediana Edad , Neovascularización Patológica/inmunología , Neovascularización Patológica/metabolismoRESUMEN
BACKGROUND/AIMS: Adipose-derived Stem Cells (ASCs) are used in Regenerative Medicine, including fat grafting, recovery from local tissue ischemia and scar remodeling. The aim of this study was to evaluate hyaluronan based gel effects on ASCs differentiation and proliferation. METHODS: Comparative analyses using high (H) and low (L) molecular weight hyaluronans (HA), hyaluronan hybrid cooperative complexes (HCCs), and high and medium cross-linked hyaluronan based dermal fillers were performed. Human ASCs were characterized by flow cytometry using CD90, CD34, CD105, CD29, CD31, CD45 and CD14 markers. Then, cells were treated for 7, 14 and 21 days with hyaluronans. Adipogenic differentiation was evaluated using Oil red-O staining and expression of leptin, PPAR-γ, LPL and adiponectin using qRT-PCR. Adiponectin was analyzed by immunofluorescence, PPAR-γ and adiponectin were analyzed using western blotting. ELISA assays for adiponectin and leptin were performed. RESULTS: HCCs highly affected ASCs differentiation by up-regulating adipogenic genes and related proteins, that were also secreted in the culture medium. H-HA and L-HA induced a lower level of ASCs differentiation. CONCLUSION: HCCs-based formulations clearly enhance adipogenic differentiation and proliferation, when compared with linear HA and cross-linked hyaluronans. Injection of HCCs in subdermal fat compartment may recruit and differentiate stem cells in adipocytes, and considerably improving fat tissue renewal.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Ácido Hialurónico/farmacología , Adipogénesis/efectos de los fármacos , Adiponectina/análisis , Adiponectina/metabolismo , Tejido Adiposo/citología , Adulto , Antígenos CD/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Ácido Hialurónico/química , Leptina/análisis , Leptina/metabolismo , Lipoproteína Lipasa/metabolismo , Microscopía Fluorescente , Persona de Mediana Edad , Peso Molecular , PPAR gamma/metabolismo , Fenotipo , Células Madre/citología , Células Madre/metabolismo , Cirugía PlásticaRESUMEN
BACKGROUND: Inflammation plays a key role in the pathogenesis of several chronic diseases. The urokinase plasminogen activator receptor (uPAR) exerts a plethora of functions in both physiological and pathological processes, including inflammation. OBJECTIVE AND DESIGN: In this study, we evaluated the anti-inflammatory effect of a novel peptide ligand of uPAR, UPARANT, in different animal models of inflammation. SUBJECTS AND TREATMENT: Rats and mice were divided in different groups (n = 5) for single or repeated administration of vehicle (9% DMSO in 0.9% NaCl), UPARANT (6, 12 and 24 mg/kg) or dexamethasone (2 mg/kg). Animals were subjected to carrageenan-induced paw oedema or zymosan-induced peritonitis. METHODS: UPARANT effects were tested on: (1) the carrageenan-induced paw oedema volume, (2) the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and the nitrite/nitrate (NOx) levels in the paw exudates, (3) cells recruitment into the peritoneal cavity after zymosan injection and (4) NOx levels in the peritoneal lavage. RESULTS: UPARANT (12 and 24 mg/kg) reduced inflammation in both experimental paradigms. Analysis of pro-inflammatory enzymes revealed that administration of UPARANT reduced iNOS, COX2 and NO over-production. CONCLUSIONS: Our study provides a solid evidence that UPARANT reduces the severity of inflammation in diverse animal models, thus representing a novel anti-inflammatory drug with potential advantages with respect to the typical steroidal agents.
Asunto(s)
Antiinflamatorios/uso terapéutico , Edema/tratamiento farmacológico , Oligopéptidos/uso terapéutico , Animales , Carragenina , Ciclooxigenasa 2/metabolismo , Dexametasona/uso terapéutico , Edema/inducido químicamente , Edema/metabolismo , Masculino , Ratones , Nitratos/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Nitritos/metabolismo , Lavado Peritoneal , Peritonitis/tratamiento farmacológico , Ratas Wistar , ZimosanRESUMEN
Thermostable phosphotriesterase-like lactonases (PLLs) from extremophile archaea, like SsoPox from Sulfolobus solfataricus, are attractive biotechnological tools with industrial applications as organophosphate decontaminants, but their manufacturing still remains an unresolved issue because of the high costs and the low production yields. In this paper, for the first time, an efficient biotechnological process for the production and purification of a recombinant, engineered PLL, SsoPox W263F, expressed in E. coli, has been set up by studying new induction strategies, by designing high cell density cultivations and a new membrane-based downstream process. In fed batches, the enzyme production was boosted of 69-fold up to 4660.0 U L-1 using galactose as inducer in the replacement of IPTG; the process was scalable from 2.5 up to 150 L. By coupling a single thermo-precipitation step and an ultrafiltration process, a total enzyme recovery of 77% with a purity grade of almost 80% was reached.
Asunto(s)
Hidrolasas de Éster Carboxílico/biosíntesis , Genes Arqueales , Organofosfatos/química , Hidrolasas de Triéster Fosfórico/biosíntesis , Sulfolobus solfataricus/genética , Técnicas de Cultivo Celular por Lotes , Medios de Cultivo/química , Descontaminación , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentación , Microbiología Industrial , Microorganismos Modificados Genéticamente , Ingeniería de Proteínas , Sulfolobus solfataricus/metabolismoRESUMEN
Cartilage tissue engineering, with in vitro expansion of autologus chondrocytes, is a promising technique for tissue regeneration and is a new potential strategy to prevent and/or treat cartilage damage (e.g., osteoarthritis). The aim of this study was (i) to investigate and compare the effects of new biotechnological chondroitin (BC) and a commercial extractive chondroitin sulfate (CS) on human chondrocytes in vitro culture; (ii) to evaluate the anti-inflammatory effects of the innovative BC compared to extractive CS. A chondrogenic cell population was isolated from human nasoseptal cartilage and in vitro cultures were studied through time-lapse video microscopy (TLVM), immunohistochemical staining and cytometry. In order to investigate the effect of BC and CS on phenotype maintainance, chondrogenic gene expression of aggrecan (AGN), of the transcriptor factor SOX9, of the types I and II collagen (COL1A1 and COL1A2), were quantified through transcriptional and protein evaluation at increasing cultivation time and passages. In addition to resemble the osteoarthritis-like in vitro model, chondrocytes were treated with IL-1ß and the anti-inflammatory activity of BC and CS was assessed using cytokines quantification by multiplex array. BC significantly enhances cell proliferation also preserving chondrocyte phenotype increasing type II collagen expression up to 10 days of treatment and reduces inflammatory response in IL-1ß treated chondrocytes respect to CS treated cells. Our results, taken together, suggest that this new BC is of foremost importance in translational medicine because it can be applied in novel scaffolds and pharmaceutical preparations aiming at cartilage pathology treatments such as the osteoarthritis. J. Cell. Biochem. 117: 2158-2169, 2016. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.
Asunto(s)
Antígenos de Diferenciación/biosíntesis , Condrocitos/metabolismo , Condroitín/química , Regulación de la Expresión Génica , Andamios del Tejido/química , Células Cultivadas , Condrocitos/citología , HumanosRESUMEN
Fucosylated chondroitin sulfate (fCS)-a glycosaminoglycan (GAG) found in sea cucumbers-has recently attracted much attention owing to its biological properties. In particular, a low molecular mass fCS polysaccharide has very recently been suggested as a strong candidate for the development of an antithrombotic drug that would be safer and more effective than heparin. To avoid the use of animal sourced drugs, here we present the chemical transformation of a microbial sourced unsulfated chondroitin polysaccharide into a small library of fucosylated (and sulfated) derivatives thereof. To this aim, a modular approach based on the different combination of only five reactions was employed, with an almost unprecedented polysaccharide branching by O-glycosylation as the key step. The library was differentiated for sulfation patterns and/or positions of the fucose branches, as confirmed by detailed 2D NMR spectroscopic analysis. These semi-synthetic polysaccharides will allow a wider and more accurate structure-activity relationship study with respect to those reported in literature to date.
Asunto(s)
Anticoagulantes/química , Sulfatos de Condroitina/química , Fucosa/química , Heparina/química , Polisacáridos/química , Pepinos de Mar/química , Animales , Anticoagulantes/farmacología , Glicosilación , Heparina/farmacología , Espectroscopía de Resonancia Magnética , Relación Estructura-ActividadRESUMEN
A semisynthetic approach to novel lipidâ A derivatives from Escherichia coli (E.â coli) lipidâ A is reported. This methodology stands as an alternative to common approaches based exclusively on either total synthesis or extraction from bacterial sources. It relies upon the purification of the lipidâ A fraction from fed-batch fermentation of E.â coli, followed by its structural modification through tailored, site-selective chemical reactions. In particular, modification of the lipid pattern and functionalization of the phosphate group as well as of the sole primary hydroxyl group were accomplished, highlighting the unusual reactivity of the molecule. Preliminary investigations of the immunostimulating activity of the new semisynthetic lipidâ A derivatives show that some of them stand out as promising, new immunoadjuvant candidates.
Asunto(s)
Lípido A/análogos & derivados , Adyuvantes Inmunológicos , Escherichia coli/química , Lípido A/químicaRESUMEN
Osteoarthritis (OA) represents a group of chronic, painful, disabling conditions affecting synovial joints. It is characterized by degeneration of articular cartilage, alterations of peri-articular and subchondral bone, low-grade synovial inflammation (synovitis). Despite OA is commonly described as a non-inflammatory disease, it is known that its progression and the subsequent increment of symptoms correlate to the production of inflammatory factors that induce the secretion of enzymes responsible for cartilage degradation. In clinical practice, to alleviate pain and stiffness, not only during acute phases but also as maintenance therapy, intra-articular injections of corticosteroids or similar drugs are used, besides it is well diffused the viscosupplementation procedure based on hyaluronan gel. There are many different products containing high molecular weight linear HA or cross-linked derivatives, however the novelty in the field consist in the hybrid cooperative complexes derived from high and low molecular weight HA through a patented processing. This technique permit to double the amount of HA delivered to the injured site without increasing the injected volume, beside in vitro assay on human chondrocytes suggested hybrid complexes as effective in the modulation of several inflammatory cytokines in joints.
RESUMEN
BACKGROUND: Recent studies have reported the roles of Hyaluronic acid (HA) chains of diverse length in wound repair, especially considering the simultaneous occurrence in vivo of both high- (H-HA) and low-molecular weight (L-HA) hyaluronan at an injury site. It has been shown that HA fragments (5 ≤ MW ≤ 20 kDa) usually trigger an inflammatory response that, on one hand, is the first signal in the activation of a repair mechanism but on the other, when it's overexpressed, it may promote unwanted side effects. The present experimental research has aimed to investigate H-HA, L-HA and of a newly developed complex of the two (H-HA/L-HA) for stability (e.g. hyaluronidases digestion), for their ability to promote wound healing of human keratinocytes in vitro and for their effect on cellular biomarker expression trends. RESULTS: Time-lapse video microscopy studies proved that the diverse HA was capable of restoring the monolayer integrity of HaCat. The H-HA/L-HA complex (0.1 and 1%w/v) proved faster in regeneration also in co-culture scratch test where wound closure was achieved in half the time of H-HA stimulated cells and 2.5-fold faster than the control. Gene expression was evaluated for transformation growth factor beta 1 (TGF-ß1) proving that L-HA alone increased its expression at 4 h followed by restoration of similar trends for all the stimuli. Depending on the diverse stimulation (H-HA, L-HA or the complex), metalloproteinases (MMP-2, -9, -13) were also modulated differently. Furthermore, type I collagen expression and production were evaluated. Compared to the others, persistence of a significant higher expression level at 24 h for the H-HA/L-HA complex was found. CONCLUSIONS: The outcomes of this research showed that, both at high and low concentrations, hybrid complexes proved to perform better than HA alone thus suggesting their potential as medical devices in aesthetic and regenerative medicine.
Asunto(s)
Ácido Hialurónico/farmacología , Cicatrización de Heridas/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Técnicas de Cocultivo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Humanos , Ácido Hialurónico/química , Ácido Hialurónico/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Metaloproteinasa 13 de la Matriz/genética , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Microscopía por Video , Peso Molecular , Streptococcus/metabolismo , Imagen de Lapso de Tiempo , Transcriptoma/efectos de los fármacos , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo , ViscosidadRESUMEN
Chemical O-glycosylation of polysaccharides is an almost unexplored reaction. This is mainly due to the difficulties in derivatizing such complex biomacromolecules in a quantitative manner and with a fine control of the obtained structural parameters. In this work, chondroitin raw material from a microbial source was chemo- and regioselectively protected to give two polysaccharide intermediates, that acted in turn as glycosyl acceptors in fucosylation reactions. Further manipulations on the fucosylated polysaccharides, including multiple de-O-benzylation and sulfation, furnished for the first time nonanimal sourced fucosylated chondroitin sulfates (fCSs)-polysaccharides obtained so far exclusively from sea cucumbers (Echinoidea, Holothuroidea) and showing several very interesting biological activities. A semisynthetic fCS was characterized from a structural point of view by means of 2D-NMR techniques, and preliminarily assayed in an anticoagulant test.
Asunto(s)
Sulfatos de Condroitina/síntesis química , Sulfatos de Condroitina/farmacología , Animales , Anticoagulantes/síntesis química , Anticoagulantes/química , Anticoagulantes/farmacología , Sulfatos de Condroitina/química , Imagen por Resonancia Magnética , Estructura MolecularRESUMEN
BACKGROUND: Vaginal lactic acid bacteria defend the host against pathogens through a combination of competitive exclusion, competition for nutrients, production of antimicrobial substances and through the activation of the immune system. A new human isolate named Lactobacillus crispatus L1 was characterized in this work, and a preliminary evaluation of its probiotic potential is described together with a process to obtain a high productivity of viable biomass. RESULTS: In a simulated digestion process 1.8â 10(10) cellsâml(-1) survived the gastric environment with 80% viability, without being affected by small intestine juices. Experiments on six different C sources were performed to analyze growth and organic acids production and, glucose, provided the best performances. A microfiltration strategy was exploited to improve the cellular yield in 2 L-fermentation processes, reaching 27 g · l(-1) of dry biomass. Moreover, L. crispatus L1 demonstrated a greater stability to high concentrations of lactic acid, compared to other lactobacilli. The specific L. crispatus L1 exopolysaccharide was purified from the fermentation broth and characterized by NMR showing structural features and similarity to exopolysaccharides produced by pathogenic strains. Live L. crispatus L1 cells strongly reduced adhesion of a yeast pathogenic strain, Candida albicans in particular, in adherence assays. Interestingly a higher expression of the human defensin HBD-2 was also observed in vaginal cells treated with the purified exopolysaccharide, indicating a possible correlation with C. albicans growth inhibition. CONCLUSIONS: The paper describes the evaluation of L. crispatus L1 as potential vaginal probiotic and the fermentation processes to obtain high concentrations of viable cells.
Asunto(s)
Lactobacillus/química , Lactobacillus/crecimiento & desarrollo , Polisacáridos Bacterianos/aislamiento & purificación , Probióticos/química , Probióticos/aislamiento & purificación , Antibiosis , Candida albicans/fisiología , Ácidos Carboxílicos/metabolismo , Adhesión Celular , Recuento de Células , Femenino , Glucosa/metabolismo , Humanos , Lactobacillus/efectos de los fármacos , Lactobacillus/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Interacciones Microbianas , Viabilidad Microbiana/efectos de los fármacos , Jugo Pancreático/metabolismo , Polisacáridos Bacterianos/química , Vagina/microbiologíaRESUMEN
Chikungunya virus (CHIKV), a mosquito-borne arthrogenic Alphavirus, causes an acute febrile illness in humans, that is, accompanied by severe joint pains. In many cases, the infection leads to persistent arthralgia, which may last for weeks to several years. The re-emergence of this infection in the early 2000s was exemplified by numerous outbreaks in the eastern hemisphere. Since then, the virus is rapidly spreading. Currently, no drugs have been approved or are in development for the treatment of CHIKV, which makes this viral infection particularly interesting for academic medicinal chemistry efforts. Several molecules have already been identified that inhibit CHIKV replication in phenotypic virus-cell-based assays. One of these is arbidol, a molecule that already has been licensed for the treatment of influenza A and B virus infections. For structural optimization, a dedicated libraries of 43 indole-based derivatives were evaluated leading to more potent analogues (IIIe and IIIf) with anti-chikungunya virus (CHIKV) activities higher than those of the other derivatives, including the lead compound, and with a selective index of inhibition 13.2 and 14.6, respectively, higher than that of ARB (4.6).
Asunto(s)
Antivirales/química , Antivirales/farmacología , Fiebre Chikungunya/tratamiento farmacológico , Virus Chikungunya/efectos de los fármacos , Indoles/química , Indoles/farmacología , Replicación Viral/efectos de los fármacos , Animales , Virus Chikungunya/fisiología , Chlorocebus aethiops , Humanos , Relación Estructura-Actividad , Células VeroRESUMEN
Streptomyces roseochromogenes is able to hydroxylate steroid compounds in different positions of their cycloalkane rings thanks to a cytochrome P-450 multi-enzyme complex. In this paper, the hydroxylation of the hydrocortisone in the 16α position, performed by bacterial whole cells, was investigated in both shake flask and fermentation conditions; the best settings for both cellular growth and transformation reaction were studied by investigating the optimal medium composition, the kinetic of conversion, the most suitable substrate concentration and the preferred addition timing. Using newly formulated malt extract- and yeast extract-based media, a 16α-hydrohydrocortisone concentration of 0.2 ± 0.01 g L(-1) was reached in shake flasks. Batch experiments in a 2-L fermentor established the reproducibility and robustness of the biotransformation, while a pulsed batch fermentation strategy allowed the production to increase up to 0.508 ± 0.01 g L(-1). By-product formation was investigated, and two new derivates of the hydrocortisone obtained during the bacterial transformation reaction and unknown so far, a C-20 hydroxy derivate and a C-21 N-acetamide one, were determined by NMR analyses.