Search results for: in vitro rumen fermentation

Authors: Joan O. Ogundele, Aladesanmi A. Oshodi, Adekunle I. Amoo

Abstract:

Abstract In-vitro multi-enzyme protein digestibility (IVMPD) and some anti-nutritional factors (ANF) of five melon (egusi) seed flours (MSF) and their protein isolates (PI) were carried out. Their PI have potentials comparable to that of soya beans. It is important to know the IVMPD and ANF of these protein sources as to ensure their safety when adapted for use as alternate protein sources to substitute for cow milk, which is relatively expensive in Nigeria. Standard methods were used to produce PI of Citrullus colocynthis, Citrullus vulgaris, African Wine Kettle gourd (Lageneria siceraria I), Basket Ball gourd (Lagenaria siceraria II) and Bushel Giant Gourd (Lageneria siceraria III) seeds and to determine the ANF and IVMPD of the MSF and PI unheated and at 37oC. Multi-enzymes used were trypsin, chymotrypsin and peptidase. IVMPD of MSF ranged from (70.67±0.70) % (C. vulgaris) to (72.07± 1.79) % (L.siceraria I) while for their PI ranged from 74.33% (C.vulgaris) to 77.55% (L.siceraria III). IVMPD of the PI were higher than those of MSF. Heating increased IVMPD of MSF with average value of 79.40% and those of PI with average of 84.14%. ANF average in MSF are tannin (0.11mg/g), phytate (0.23%). Differences in IVMPD of MSF and their PI at different temperatures may arise from processing conditions that alter the release of amino acids from proteins by enzymatic processes. ANF in MSF were relatively low, but were found to be lower in the PI, therefor making the PI safer for human consumption as an alternate source of protein.

Keywords: Anti-nutrients, Enzymatic protein digestibility, Melon (egusi)., Protein Isolates.

Procedia PDF Downloads 95

Authors: Alok Raghav, Jamal Ahmad

Abstract:

Background: Serum albumin glycation and advanced glycation end products (AGE) formation correlates in diabetes and its associated complications. Extensive modified human serum albumin is used to study the biochemical, electrochemical and functional properties in hyperglycemic environment with relevance to diabetes. We evaluate Spectroscopic, side chain modifications, amino acid analysis, biochemical and functional group properties in four glucose modified samples. Methods: A series four human serum albumin samples modified with glucose was characterized in terms of amino acid analysis, spectroscopic properties and side chain modifications. The diagnostic technique employed incorporates UV Spectroscopy, Fluorescence Spectroscopy, biochemical assays for side chain modifications, amino acid estimations, electrochemical and optical characterstic of glycated albumin. Conclusion: Glucose modified human serum albumin confers AGEs formation alters biochemical, electrochemical, optical, and functional property that depend on the reactivity of glucose and its concentration used for in-vitro glycation. A biochemical, electrochemical, optical, and functional characterization of modified albumin in-vitro produced AGE product that will be useful to interpret the complications and pathophysiological significance in diabetes.

Keywords: human serum albumin, glycated albumin, adavanced glycation end products, associated pathologies

Procedia PDF Downloads 387

Authors: Jong Il Rhee

Abstract:

The polypeptide representing the mature part of human BMP-7 was cloned and efficiently expressed in Escherichia coli and Bacillus subtilis, which had a clear band for hBMP-7, a homodimeric protein with an apparent molecular weight of 15.4 kDa. Recombinant E.coli produced 111 pg hBMP-7/mg of protein hBMP-7 through IPTG induction. Recombinant B. subtilis also produced 350 pg hBMP-7/ml of culture medium. The hBMP-7 was purified in 2 steps using an FPLC system with an ion exchange column and a gel filtration column. The hBMP-7 produced in this work also stimulated the alkaline phosphatase (ALP) activity in a dose-dependent manner, i.e. 2.5- and 8.9-fold at 100 and 300 ng hBMP-7/ml, respectively, and showed intact biological activity.

Keywords: B. subtilis, E. coli, fermentation, hBMP-7

Procedia PDF Downloads 411

Authors: A. Kazbekova, A. Kudaibergenov, G. Atazhanova, S. Adekenov

Abstract:

In recent years, it achieved some progress such a direction as to study the possibility of correlation between different types of biological activity. In particular, in our work, we consider questions such as: the impact of the qualitative composition of total substances in the example of plant extracts on antioxidant and antiradical activity, the presents of correlation between these types of activity, etc. It is known that there is a relationship between the values of optical density of working solutions of extracts and corresponding bioactivity in vitro, in particular, the antioxidant and hepatoprotective effects. In this study, we have identified that among some studied species of wormwood (Artemisia viridis Wild, Artemisia jacutica Drob, Artemisia annua L, Artemisia siversiana Wild, Artemisia adamsii Bess, Artemisia tianschanica, Artemisia obtusiloba Ledeb., Artemisia heptopotamica), as well as extracts of Inula caspica, Аjania tenuifolia, Abies sibirica, Galatella songorica, Mentha asiatica and Thymus mugodzharicus it was identified that the highest content of polyphenol compounds is in Thymus mugodzharicus. At the same time, we determined the antioxidant and antiradical activity, which was the highest for the Thymus mugodzharicus. Butylhydroxyanisole and ascorbic acid were used as comparison substances. Also, it was established that antioxidant and anti-radical activities depend on the concentration of the of all investigated samples. Based on obtained data, we believe that the extract of Thymus mugodzharicus can be recommended for further study on the antioxidant and antiradical activity in vivo, as well as the opportunity of this sample to demonstrate hepatoprotective effect. The study was sponsored by SANTO academic program.

Keywords: in vitro, in vivo, antioxidant, hepatoprotective effect

Procedia PDF Downloads 306

Authors: Yanin Hosakun, Sujitra Wongkasemjit, Thanyalak Chaisuwan

Abstract:

Carbon dioxide removal from natural gas is an important process because the existence of carbon dioxide in natural gas contributes to pipeline corrosion, reduces the heating value, and takes up volume in the pipeline. In this study, bacterial cellulose was chosen for the CO2/CH4 gas separation membrane due to its unique structure and prominent properties. Additionally, it can simply be obtained by culturing the bacteria so called “Acetobacter xylinum” through fermentation of coconut juice. Bacterial cellulose membranes with and without silver ions were prepared and studied for the separation performance of CO2 and CH4.

Keywords: bacterial cellulose, CO2, CH4 separation, membrane, nata de coco

Procedia PDF Downloads 235

Authors: Narine Ghazaryan, Joana Catarina Macedo, Sara Vaz, Naira Ayvazyan, Elsa Logarinho

Abstract:

Macrovipera lebetina obtusa (MLO) is a poisonous snake in Armenia. Obtustatin represents the shortest known monomeric disintegrin, isolated from the snake venom of MLO, and is known to specifically inhibit α1β1 integrin. Its oncostatic effect is due to the inhibition of angiogenesis, which likely arises from α1β1 integrin inhibition in the endothelial cells. To explore the therapeutic potential of the MLO snake venom and obtustatin, we studied activity of obtustatin and MLO venom in vitro, by testing their efficacy in human dermal microvascular endothelial cells (HMVEC-D) and in vivo, using chick embryo chorioallantoic membrane assay (CAM assay). Our in vitro results showed that obtustatin in comparison with MLO venom did not exhibit cytotoxic activity in HMVEC-D cells in comparison to MLO venom. But in vivo results have shown that 4µg /embryo (90 µM) of obtustatin inhibited angiogenesis induced by FGF2 by 17% while MLO snake venom induced 22% reduction of the angiogenic index. The concentration of obtustatin in the crude MLO venom was 0.3 nM, which is 300.000 times less than the concentration of the obtustatin itself. Given this enormous difference in concentration, it is likely that some components of the crude venom contribute to the observed anti-angiogenic effect. Hypotheses will be ascertained to justify this action: components in the MLO venom may increase obtustatin efficacy or have independent but synergic anti-angiogenic activities.

Keywords: angiogenesis, alpa1 beta 1 integrin, Macrovipera lebetina obtusa, obtustatin

Procedia PDF Downloads 174

Authors: Shabnam Alizadeh, Hatice Dumanoglu

Abstract:

Plant tissue culture is a substantial plant propagation technique for mass clonal production throughout the year, regardless of time in fruit species. However, the rooting achievement must be enhanced in the difficult-to-root genotypes. Classical auxin applications in clonal propagation of these genotypes are inadequate to solve the rooting problem. Nanoparticles having different physical and chemical properties from bulk material could enhance the rooting success of controlled release of these substances when loaded with auxin due to their ability to reach the active substance up to the target cells as a carrier system.The purpose of this study is to investigate the effects of zinc oxide nanoparticles loaded with indole-3-acetic acid (IAA-nZnO) and indole-3-butyric acid (IBA-nZnO) on in vitro rooting of microcuttings in a difficult-to-root apple genotype (Malus domestica Borkh.). Rooting treatments consisted of IBA or IAA at concentrations of 0.5, 1.0, 2.0, 3.0 mg/L; nZnO, IAA-nZnO and IBA-nZnO at doses of 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 mg/L were used. All components were added to the Murashige and Skoog (MS) basal medium at strength ½ with 2% sucrose and 0.7% agar before autoclaving. In the study, no rooting occurred in control and nZnO applications. Especially, 1.0 mg/L and 2.0 mg/L IBA-nZnO nanoparticle applications (containing 0.5 mg/L and 0.9 mg/L IBA), respectively with rooting rates of 40.3% and 70.4%, rooting levels of 2.0±0.4 and 2.3±0.4, 2.6±0.7 and 2.5±0.6 average root numbers and 20.4±1.6 mm and 20.2±3.4 mm average root lengths put forward as effective applications.

Keywords: Auxin, Malus, nanotechnology, zinc oxide nanoparticles

Procedia PDF Downloads 132

Authors: Alija Uzunovic, Sasa Pilipovic, Aida Sapcanin, Zahida Ademovic, Berina Pilipović

Abstract:

Capsaicin is a naturally occurring alkaloid extracted from capsicum fruit extracts of different of Capsicum species. It has been employed topically to treat many diseases such as rheumatoid arthritis, osteoarthritis, cancer pain and nerve pain in diabetes. The high degree of pre-systemic metabolism of intragastrical capsaicin and the short half-life of capsaicin by intravenous administration made topical application of capsaicin advantageous. In this study, we have evaluated differences in the dissolution characteristics of capsaicin patch 11 mg (purchased from market) at different dissolution rotation speed. The proposed patch area is 308 cm2 (22 cm x 14 cm; it contains 36 µg of capsaicin per square centimeter of adhesive). USP Apparatus 5 (Paddle Over Disc) is used for transdermal patch testing. The dissolution study was conducted using USP apparatus 5 (n=6), ERWEKA DT800 dissolution tester (paddle-type) with addition of a disc. The fabricated patch of 308 cm2 is to be cut into 9 cm2 was placed against a disc (delivery side up) retained with the stainless-steel screen and exposed to 500 mL of phosphate buffer solution pH 7.4. All dissolution studies were carried out at 32 ± 0.5 °C and different rotation speed (50± 5; 100± 5 and 150± 5 rpm). 5 ml aliquots of samples were withdrawn at various time intervals (1, 4, 8 and 12 hours) and replaced with 5 ml of dissolution medium. Withdrawn were appropriately diluted and analyzed by reversed-phase liquid chromatography (RP-LC). A Reversed Phase Liquid Chromatography (RP-LC) method has been developed, optimized and validated for the separation and quantitation of capsaicin in a transdermal patch. The method uses a ProntoSIL 120-3-C18 AQ 125 x 4,0 mm (3 μm) column maintained at 600C. The mobile phase consisted of acetonitrile: water (50:50 v/v), the flow rate of 0.9 mL/min, the injection volume 10 μL and the detection wavelength 222 nm. The used RP-LC method is simple, sensitive and accurate and can be applied for fast (total chromatographic run time was 4.0 minutes) and simultaneous analysis of capsaicin and dihydrocapsaicin in a transdermal patch. According to the results obtained in this study, we can conclude that the relative difference of dissolution rate of capsaicin after 12 hours was elevated by increase of dissolution rotation speed (100 rpm vs 50 rpm: 84.9± 11.3% and 150 rpm vs 100 rpm: 39.8± 8.3%). Although several apparatus and procedures (USP apparatus 5, 6, 7 and a paddle over extraction cell method) have been used to study in vitro release characteristics of transdermal patches, USP Apparatus 5 (Paddle Over Disc) could be considered as a discriminatory test. would be able to point out the differences in the dissolution rate of capsaicin at different rotation speed.

Keywords: capsaicin, in vitro, patch, RP-LC, transdermal

Procedia PDF Downloads 211

Authors: Joulié Aurélien, Jourdain Elsa, Bailly Xavier, Gasqui Patrick, Yang Elise, Leblond Agnès, Rousset Elodie, Sidi-Boumedine Karim

Abstract:

Q fever is a worldwide zoonosis caused by the gram-negative obligate intracellular bacterium Coxiella burnetii. Following the recent outbreaks in the Netherlands, a hyper virulent clone was found to be the cause of severe human cases of Q fever. In livestock, Q fever clinical manifestations are mainly abortions. Although the abortion rates differ between ruminant species, C. burnetii’s virulence remains understudied, especially in enzootic areas. In this study, the infectious potential of three C. burnetii isolates collected from French farms of small ruminants were compared to the reference strain Nine Mile (in phase II and in an intermediate phase) using an in vivo (CD1 mice) model. Mice were challenged with 105 live bacteria discriminated by propidium monoazide-qPCR targeting the icd-gene. After footpad inoculation, spleen and popliteal lymph node were harvested at 10 days post-inoculation (p.i). The strain invasiveness in spleen and popliteal nodes was assessed by qPCR assays targeting the icd-gene. Preliminary results showed that the avirulent strains (in phase 2) failed to pass the popliteal barrier and then to colonize the spleen. This model allowed a significant differentiation between strain’s invasiveness on biological host and therefore identifying distinct virulence profiles. In view of these results, we plan to go further by testing fifteen additional C. burnetii isolates from French farms of sheep, goat and cattle by using the above-mentioned in vivo model. All 15 strains display distant MLVA (multiple-locus variable-number of tandem repeat analysis) genotypic profiles. Five of the fifteen isolates will bee also tested in vitro on ovine and bovine macrophage cells. Cells and supernatants will be harvested at day1, day2, day3 and day6 p.i to assess in vitro multiplication kinetics of strains. In conclusion, our findings might help the implementation of surveillance of virulent strains and ultimately allow adapting prophylaxis measures in livestock farms.

Keywords: Q fever, invasiveness, ruminant, virulence

Procedia PDF Downloads 346

Authors: Loren S. Bernal., Catalina Castillo, Carel E. Carvajal, José F. Ibla

Abstract:

Plastic pollution, specifically microplastics, has become a significant issue in aquatic ecosystems worldwide. The large amount of plastic waste carried by water tributaries has resulted in the accumulation of microplastics in water bodies. The polymer aging process caused by environmental influences such as photodegradation and chemical degradation of additives leads to polymer embrittlement and properties change that require degradation or reduction procedures in rivers. However, there is a lack of such procedures for freshwater entities that develop over extended periods. The aim of this study is evaluate the potential of Pleurotus ostreatus a fungus, in reducing lowdensity polyethylene microplastics present in freshwater samples collected from the middle basin of the Magdalena River in Colombia. The study aims to evaluate this process both in vitro and in silico by identifying the growth capacity of Pleurotus ostreatus in the presence of microplastics and identifying the most likely interactions of Pleurotus ostreatus enzymes and their affinity energies. The study follows an engineering development methodology applied on an experimental basis. The in vitro evaluation protocol applied in this study focused on the growth capacity of Pleurotus ostreatus on microplastics using enzymatic inducers. In terms of in silico evaluation, molecular simulations were conducted using the Autodock 1.5.7 program to calculate interaction energies. The molecular dynamics were evaluated by using the myPresto Portal and GROMACS program to calculate radius of gyration and Energies.The results of the study showed that Pleurotus ostreatus has the potential to degrade low-density polyethylene microplastics. The in vitro evaluation revealed the adherence of Pleurotus ostreatus to LDPE using scanning electron microscopy. The best results were obtained with enzymatic inducers as a MnSO4 generating the activation of laccase or manganese peroxidase enzymes in the degradation process. The in silico modelling demonstrated that Pleurotus ostreatus was able to interact with the microplastics present in LDPE, showing affinity energies in molecular docking and molecular dynamics shown a minimum energy and the representative radius of gyration between each enzyme and its substract. The study contributes to the development of bioremediation processes for the removal of microplastics from freshwater sources using the fungus Pleurotus ostreatus. The in silico study provides insights into the affinity energies of Pleurotus ostreatus microplastic degrading enzymes and their interaction with low-density polyethylene. The study demonstrated that Pleurotus ostreatus can interact with LDPE microplastics, making it a good agent for the development of bioremediation processes that aid in the recovery of freshwater sources. The results of the study suggested that bioremediation could be a promising approach to reduce microplastics in freshwater systems.

Keywords: bioremediation, in silico modelling, microplastics, Pleurotus ostreatus

Procedia PDF Downloads 95

Authors: Camelia Vizireanu, Daniela Istrati, Alina Georgiana Profir, Rodica Mihaela Dinica

Abstract:

Globally, there is a great interest in promoting the consumption of fruit and vegetables to improve health. Due to the content of essential compounds such as antioxidants, important amounts of fruits and vegetables should be included in the daily diet. Juices are good sources of vitamins and can also help increase overall fruit and vegetable consumption. Starting from this trend (introduction into the daily diet of vegetables and fruits) as well as the desire to diversify the range of functional products for both adults and children, a fermented juice was made using probiotic microorganisms based on root vegetables, with potential beneficial effects in the diet of children, vegetarians and people with lactose intolerance. The three vegetables selected for this study, red beet, carrot, and celery bring a significant contribution to functional compounds such as carotenoids, flavonoids, betalain, vitamin B and C, minerals and fiber. By fermentation, the functional value of the vegetable juice increases due to the improved stability of these compounds. The combination of probiotic microorganisms and vegetable fibers resulted in a nutrient-rich synbiotic product. The stability of the nutritional and sensory qualities of the obtained synbiotic product has been tested throughout its shelf life. The evaluation of the physico-chemical changes of the synbiotic drink during storage confirmed that: (i) vegetable juice enriched with honey and vegetable pulp is an important source of nutritional compounds, especially carbohydrates and fiber; (ii) microwave treatment used to inhibit pathogenic microflora did not significantly affect nutritional compounds in vegetable juice, vitamin C concentration remained at baseline and beta-carotene concentration increased due to increased bioavailability; (iii) fermentation has improved the nutritional quality of vegetable juice by increasing the content of B vitamins, polyphenols and flavonoids and has a good antioxidant capacity throughout the shelf life; (iv) the FTIR and Raman spectra have highlighted the results obtained using physicochemical methods. Based on the analysis of IR absorption frequencies, the most striking bands belong to the frequencies 3330 cm⁻¹, 1636 cm⁻¹ and 1050 cm⁻¹, specific for groups of compounds such as polyphenols, carbohydrates, fatty acids, and proteins. Statistical data processing revealed a good correlation between the content of flavonoids, betalain, β-carotene, ascorbic acid and polyphenols, the fermented juice having a stable antioxidant activity. Also, principal components analysis showed that there was a negative correlation between the evolution of the concentration of B vitamins and antioxidant activity. Acknowledgment: This study has been founded by the Francophone University Agency, Project Réseau régional dans le domaine de la santé, la nutrition et la sécurité alimentaire (SaIN), No. at Dunarea de Jos University of Galati 21899/ 06.09.2017 and by the Sectorial Operational Programme Human Resources Development of the Romanian Ministry of Education, Research, Youth and Sports trough the Financial Agreement POSDRU/159/1.5/S/132397 ExcelDOC.

Keywords: bioactive compounds, fermentation, synbiotic drink from vegetables, stability during storage

Procedia PDF Downloads 139

Authors: K. S. Hemant Yadav, H. G. Shivakumar

Abstract:

The present study investigation was to prepare and evaluate the mucoadhesive multi-particulate system for nasal drug delivery of anti-histaminic drug. Ebastine was chosen as the model drug. Drug loaded nanoparticles of Ebastine were prepared by ionic gelation method using chitosan as polymer using the drug-polymer weight ratios 1:1, 1:2, 1:3. Sodium tripolyphosphate (STPP) was used as the cross-linking agent in the range of 0.5 and 0.7% w/v. FTIR and DSC studies indicated that no chemical interaction occurred between the drug and polymers. Particle size ranged from 169 to 500 nm. The drug loading and entrapment efficiency was found to increase with increase in chitosan concentration and decreased with increase in poloxamer 407 concentration. The results of in vitro mucoadhesion carried out showed that all the prepared formulation had good mucoadhesive property and mucoadhesion increases with increase in the concentration of chitosan. The in vitro release pattern of all the formulations was observed to be in a biphasic manner characterized by slight burst effect followed by a slow release. By the end of 8 hrs, formulation F6 showed a release of only 86.9% which explains its sustained behaviour. The ex-vivo permeation of the pure drug ebastine was rapid than the optimized formulation(F6) indicating the capability of the chitosan polymer to control drug permeation rate through the sheep nasal mucosa. The results indicated that the mucoadhesive nanoparticulate system can be used for the nasal delivery of antihistaminic drugs in an effective manner.

Keywords: nasal, nanoparticles, ebastine, anti-histaminic drug, mucoadhesive multi-particulate system

Procedia PDF Downloads 406

Authors: Callistus I. Iheme, Kenneth E. Asika, Emmanuel I. Ugwor, Chukwuka U. Ogbonna, Ugonna H. Uzoka, Nneamaka A. Chiegboka, Chinwe S. Alisi, Obinna S. Nwabueze, Amanda U. Ezirim, Judeanthony N. Ogbulie

Abstract:

Antimicrobial resistance (AMR) is a major threat to the global health sector. Zinc oxide nanocomposites (ZnONCs), composed of zinc oxide nanoparticles and phytochemicals from Azadirachta indica aqueous leaf extract, were assessed for their physico-chemicals, antioxidant, in silico, and in vitro antimicrobial properties on multidrug-resistant Escherichia coli enzymes. Gas chromatography coupled with mass spectroscope (GC-MS) analysis on the ZnONCs revealed the presence of twenty volatile phytochemical compounds, among which is scoparone. Characterization of the ZnONCs was done using Ultraviolet-visible spectroscopy (UV-vis), energy dispersive spectroscopy (EDX), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and x-ray diffractometer (XRD). The results showed arrays of ZnONCs nanorods with maximal absorption wavelengths of 320 nm and 350 nm and thermally stable at the temperature range of 423.77 to 889.69 ℃. In vitro, the study assessed the dehydrogenase inhibitory properties of the ZnONCs, a conjugate of ZnONCs and ampicillin (ZnONCs-amp), the aqueous leaf extract of A. indica, and ampicillin (standard drug). The findings revealed that at the concentration of 500 μm/mL, 57.89 % of the enzyme activities were inhibited by ZnONCs compared to 33.33 % and 21.05 % of the standard drug (Ampicillin), and the aqueous leaf extract of the A. indica respectively. The inhibition of the enzyme activities by the ZnONCs at 500 μm/mL was further enhanced to 89.74 % by conjugating with Ampicillin. In silico study on the ZnONCs revealed scoparone as the most viable competitor of nicotinamide adenine dinucleotide (NAD+) for the coenzyme binding pocket on E. coli malate and histidinol dehydrogenase. The antioxidant analysis using DPPH indicated that ZnONCs exhibited lower reducing power when compared to the standard (garlic acid). From the findings, it can be concluded that the scoparone components of the nanocomposites in synergy with the zinc oxide nanoparticles inhibited E. coli malate and histidine dehydrogenase by competitively binding to the NAD+ pocket and that the conjugation of the ZnONCs with ampicillin further enhanced the antimicrobial efficiency of the nanocomposite against multidrug-resistant E. coli.

Keywords: antimicrobial resistance (AMR), dehydrogenase activities, E. coli, zinc oxide nanocomposites

Procedia PDF Downloads 12

Authors: Brajesh Tiwari, Yuvraj Dangi, Abhishek Jain, Ashok Jain

Abstract:

The purpose of the investigation was to evaluate the potential of sphingosomes as nanoscale drug delivery units for site-specific delivery of anti-cancer agents. Doxorubicin Hydrochloride (DOX) was selected as a model anti-cancer agent. Sphingosomes were prepared and loaded with DOX and optimized for size and drug loading. The formulations were characterized by Malvern zeta-seizer and Transmission Electron Microscopy (TEM) studies. Sphingosomal formulations were further evaluated for in-vitro drug release study under various pH profiles. The in-vitro drug release study showed an initial rapid release of the drug followed by a slow controlled release. In vivo studies of optimized formulations and free drug were performed on albino rats for comparison of drug plasma concentration. The in- vivo study revealed that the prepared system enabled DOX to have had enhanced circulation time, longer half-life and lower elimination rate kinetics as compared to free drug. Further, it can be interpreted that the formulation would selectively enter highly porous mass of tumor cells and at the same time spare normal tissues. To summarize, the use of sphingosomes as carriers of anti-cancer drugs may prove to be a fascinating approach that would selectively localize in the tumor mass, increasing the therapeutic margin of safety while reducing the side effects associated with anti-cancer agents.

Keywords: sphingosomes, anti-cancer, doxorubicin, formulation

Procedia PDF Downloads 285

Authors: Khaled M. Khleifat

Abstract:

Pseudomonas aeruginosa was isolated from infected burn patients and characterized by standard biochemical tests. The in vitro copper uptake was compared between this isolated pathogenic strain and two non-pathogenic control strains of Gram-positive bacteria Bacillusthuringiensis strain Israelisas well as Gram-negative bacteria Enterobacter aerogenes. Maximum copper uptake of 470 ppm/g biomass was obtained by P. aeruginosa strain, while the control strains B. thuringiensis and Enterobacter aerogenes had copper uptake of 350 and 383 ppm/g biomass, respectively. However, the lowest copper uptake (60 ppm/g biomass) was observed with another control the saprophytic strain Pseudomonas (Shewanella) putrefaciens. A further investigation regarding the effect of copper toxicity on bacterial growth, gave an MIC score of 600 ppm for P. aeruginosa strain compared to 460 and 300 ppm for the two Gram positive and Gram negative control strains, respectively. In tandem with these in vitro findings, blood analysis on burn patients infected with P. aeruginosa has indicated a selective decrease of copper (hypocupremia) and ceruloplasmin plasma levels. The iron metabolism was also affected by this copper deprivation leading to a similar decrease in plasma levels of PCV, iron, total iron-binding capacity, and transferrin. All these hematological changes were significantly different (P < 0.05) from the matched group of non-infected burn patients. The observed hypocupremia in infected burn patients was attributed to demanding scavenger ability by P. aeruginosa strain for the copper of plasma.

Keywords: Pseudomonas aeruginosa, hypocupremia, correlation, PCV

Procedia PDF Downloads 297

Authors: Friday G. Okibe, Edit B. Agbaji, Victor O. Ajibola, Christain C. Onoyima

Abstract:

Controlled drug delivery systems reduce dose-dependent toxicity associated with potent drugs, including anticancer drugs. In this research, hydroxyapatite (HA) and hydroxyapatite-sodium alginate nanocomposites (HASA) were successfully prepared and characterized using Fourier Transform Infrared spectroscopy (FTIR) and Scanning Electron Microscopy (SEM). The FTIR result showed absorption peaks characteristics of pure hydroxyapatite (HA), and also confirmed the chemical interaction between hydroxyapatite and sodium alginate in the formation of the composite. Image analysis from SEM revealed nano-sized hydroxyapatite and hydroxyapatite-sodium alginate nanocomposites with irregular morphologies. Particle size increased with the formation of the nanocomposites relative to pure hydroxyapatite, with no significant change in particles morphologies. Drug loading and in-vitro drug release study were carried out using synthetic body fluid as the release medium, at pH 7.4 and 37 °C and under perfect sink conditions. The result shows that drug loading is highest for pure hydroxyapatite and decreased with increasing quantity of sodium alginate. However, the release study revealed that HASA-5%wt and HASA-20%wt presented better release profile than pure hydroxyapatite, while HASA-33%wt and HASA-50%wt have poor release profiles. This shows that Methotrexate-loaded hydroxyapatite-sodium alginate if prepared under optimal conditions is a potential carrier for effective delivery of Methotrexate.

Keywords: drug-delivery, hydroxyapatite, methotrexate, nanocomposites, sodium alginate

Procedia PDF Downloads 259

Authors: Adria Rae Abigail R. Eda, Arvin C. Diesmos, Vance T. Vredenburg, Merab A. Chan

Abstract:

Mitigating strategies using symbiotic cutaneous bacteria is one of the major concerns in the conservation of amphibian population. Batrachochytrium dendrobatidis is the causative agent of chytridiomycosis associated with mass mortality and amphibian extinctions worldwide. In the Philippines, there is a lack of study on the cutaneous bacteria of Philippine amphibians that may have beneficial effects to ward off the deadly fungal infection. In this study, cutaneous bacteria from frogs were isolated and examined for anti-B. dendrobatidis activity. Eight species of frogs were collected at Mt. Palay-palay Mataas na Gulod National Park in Cavite, a site positive for the presence of B. dendrobatidis. Bacteria were isolated from the skin of frogs by swabbing the surfaces of the body and inoculated in Reasoner´s 2A (R2A) agar. Isolated bacteria were tested for potential inhibitory properties against B. dendrobatidis through zoospore inhibition assay. Results showed that frog cutaneous bacteria significantly inhibited the growth of B. dendrobatidis in vitro. By means of 16S rRNA gene primers, the anti-B. dendrobatidis bacteria were identified to be Enterobacter sp., Alcaligenes faecalis and Pseudomonas sp. Cutaneous bacteria namely Enterobacter sp. (isolates PLd33 and PCv4) and Pseudomonas (isolate PLd31) remarkably cleared the growth of B. dendrobatidis zoospore in 1% tryptone agar. Therefore, frog cutaneous bacteria inhibited B. dendrobatidis in vitro and could possibly contribute to the immunity and defense of frogs against the lethal chytridiomycosis.

Keywords: Batrachochytrium dendrobatidis, cutaneous bacteria, frogs, zoospore inhibition assay

Procedia PDF Downloads 434

Authors: Hamidreza Khodaei, Ali Daryabeigi Zand

Abstract:

Conjugated linoleic acid (CLA) is a mixture of isomers of linoleic acid. Despite the fact that 28 different isomers of CLA have already been identified, but the main isomer found in natural diets more than ninety percent CLA on intake of food constitutes demonstrates. CLA is known to be a substance that readily available by rumen microorganisms in some ruminants such as cattle and sheep would likely be made. The main objective of this research was to evaluate the impacts of CLA on lipid metabolism and enhanced fat around the muscle durability by reducing the process of oxidation. In order to implement this research, 80 female mice of the Balb/C, with 55 days of age were employed in the experiment. Treatments include various levels of CLA. Over the course of this study blood samples was also taken from the tail vein of the studied mice. Some other relevant parameters such as serum concentrations of triglycerides, total cholesterol, LDL, HDL and liver enzymes were also determined. The oxidative stability of fats TBARS technique was investigated at different intervals. The findings of the research were analyzed by statistical software of SAS 98. The results, CLA had no significant effect on liver enzymes (P > 0.05). However, it showed a statistically significant impact on triglycerides and total cholesterol. Ratio of LDL to HDL declined remarkably. Histological studies demonstrated reduced accumulation of fat in the tissues surrounding muscles.

Keywords: conjugated linoleic acid, fat metabolism, fat retention, oxidation process

Procedia PDF Downloads 181

Authors: Rouzbeh Jafari, Joe Nava

Abstract:

This study includes learnings from an engineering practice normally performed on large scale biohydrogen processes. If properly scale-up is done, biohydrogen can be a reliable pathway for biowaste valorization. Most of the studies on biohydrogen process development have used model feedstock to investigate process key performance indicators (KPIs). This study does not intend to compare different technologies with model feedstock. However, it reports economic drivers and technical challenges which help in developing a road map for expanding biohydrogen economy deployment in Canada. BBA is a consulting firm responsible for the design of hydrogen production projects. Through executing these projects, activity has been performed to identify, register and mitigate technical drawbacks of large-scale hydrogen production. Those learnings, in this study, have been applied to the biohydrogen process. Through data collected by a comprehensive literature review, a base case has been considered as a reference, and several case studies have been performed. Critical parameters of the process were identified and through common engineering practice (process design, simulation, cost estimate, and life cycle assessment) impact of these parameters on the commercialization risk matrix and class 5 cost estimations were reported. The process considered in this study is food waste and woody biomass dark fermentation. To propose a reliable road map to develop a sustainable biohydrogen production process impact of critical parameters was studied on the end-to-end process. These parameters were 1) feedstock composition, 2) feedstock pre-treatment, 3) unit operation selection, and 4) multi-product concept. A couple of emerging technologies also were assessed such as photo-fermentation, integrated dark fermentation, and using ultrasound and microwave to break-down feedstock`s complex matrix and increase overall hydrogen yield. To properly report the impact of each parameter KPIs were identified as 1) Hydrogen yield, 2) energy consumption, 3) secondary waste generated, 4) CO2 footprint, 5) Product profile, 6) $/kg-H2 and 5) environmental impact. The feedstock is the main parameter defining the economic viability of biohydrogen production. Through parametric studies, it was found that biohydrogen production favors feedstock with higher carbohydrates. The feedstock composition was varied, by increasing one critical element (such as carbohydrate) and monitoring KPIs evolution. Different cases were studied with diverse feedstock, such as energy crops, wastewater slug, and lignocellulosic waste. The base case process was applied to have reference KPIs values and modifications such as pretreatment and feedstock mix-and-match were implemented to investigate KPIs changes. The complexity of the feedstock is the main bottleneck in the successful commercial deployment of the biohydrogen process as a reliable pathway for waste valorization. Hydrogen yield, reaction kinetics, and performance of key unit operations highly impacted as feedstock composition fluctuates during the lifetime of the process or from one case to another. In this case, concept of multi-product becomes more reliable. In this concept, the process is not designed to produce only one target product such as biohydrogen but will have two or multiple products (biohydrogen and biomethane or biochemicals). This new approach is being investigated by the BBA team and the results will be shared in another scientific contribution.

Keywords: biohydrogen, process scale-up, economic evaluation, commercialization uncertainties, hydrogen economy

Procedia PDF Downloads 84

Authors: Parnaz Boodagh, Danila Vella, Antonio Damore, Laura Modica De Mohac, Sang-Ho Ye, Garret Coyan, Gaetano Burriesci, William Wagner, Federica Cosentino

Abstract:

The use of cardiac patches is among the main therapeutic solution for cardiovascular diseases, a leading mortality cause in the world with an increasing trend, responsible of 19 millions deaths in 2020. Several classes of biomaterials serve that purpose, both of synthetic origin and biological derivation, and many bioengineered treatment alternatives were proposed to satisfy two main requirements, providing structural support and promoting tissue remodeling. The objective of this paper is to compare the mechanical properties and the characterization of four cardiac patches: the Adeka, PhotoFix, CorPatch, and CardioCel patches. In vitro and in vivo tests included: biaxial, uniaxial, ball burst, suture retention for mechanical characterization; 2D surface topography, 3D volume and microstructure, and histology assessments for structure characterization; in vitro test to evaluate platelet deposition, calcium deposition, and macrophage polarization; rat right ventricular outflow tract (RVOT) models at 8- and 16-week time points to characterize the patch-host interaction. Lastly, the four patches were used to produce four stented aortic valve prosthesis, subjected to hydrodynamic assessment as well as durability testing to verify compliance with the standard ISO.

Keywords: cardiac patch, cardiovascular disease, cardiac repair, blood contact biomaterial

Procedia PDF Downloads 133

Authors: Abdul Karim Zulkarnain, Marchaban, Subagus Wahyono, Ratna Asmah Susidarti

Abstract:

Mahkota Dewa contains phalerin which has activity as sun screen. In this study, 13 formulations of cream oil in water (o/w) were prepared and tested for their physical characteristics. The physical characteristics were then used for determining the optimum formula. This study aimed to explore the physical stability of optimized formulation of cream, its sun protecting factor (SPF) values using in vitro and in vivo tests. The optimum formula of o/w cream were prepared based on Simplex Lattice Design (LSD) method using software Design Expert®. The formulation of o/w cream were varied based on the proportion of cetyl alcohol, mineral oil and tween 80. The difference of physical characteristic of optimum and predicted formula was tested using t-test with significant level of 95%. The optimum formula of o/w cream was the formula which consists of cetyl alcohol 9.71%, mineral oil, 29%, and tween 80 3.29. Based on t-test, there was no significant difference of physical characteristics of optimum and predicted formulation. Viscosity, spread power, adhesive power, and separation volume ratio of o/w at week 0-4 were relatively stable. The o/w creams were relatively stable at extreme temperature. The o/w creams from mahkota dewa, phalerin, and benzophenone have SPF values of 21.32, 33.12, and 42.49, respectively. The formulas did not irritate the skin based on in vivo test.

Keywords: cream, stability, In vitro, In vivo

Procedia PDF Downloads 212

Authors: Katarzyna Drela, Miroslaw Wielgos, Mikolaj Wrobel, Barbara Lukomska

Abstract:

Mesenchymal stem cells (MSCs) have a great potential in regenerative medicine. Since the initial number of isolated MSCs is limited, in vitro propagation is often required to reach sufficient numbers of cells for therapeutic applications. During long-term culture MSCs may undergo genetic or epigenetic alterations that subsequently increase the probability of spontaneous malignant transformation. Thus, factors that influence genomic stability of MSCs following long-term expansions need to be clarified before cultured MSCs are employed for clinical application. The aim of our study was to investigate the potential for spontaneous transformation of human neonatal cord blood (HUCB-MSCs) and adult bone marrow (BM-MSCs) derived MSCs. Materials and Methods: HUCB-MSCs and BM-MSCs were isolated by standard Ficoll gradient centrifugations method. Isolated cells were initially plated in high density 106 cells per cm2. After 48 h medium were changed and non-adherent cells were removed. The malignant transformation of MSCs in vitro was evaluated by morphological changes, proliferation rate, ability to enter cell senescence, the telomerase expression and chromosomal abnormality. Proliferation of MSCs was analyzed with WST-1 reduction method and population doubling time (PDT) was calculated at different culture stages. Then the expression pattern of genes characteristic for mesenchymal or epithelial cells, as well as transcriptions factors were examined by RT-PCR. Concomitantly, immunocytochemical analysis of gene-related proteins was employed. Results: Our studies showed that MSCs from all bone marrow isolations ultimately entered senescence and did not undergo spontaneous malignant transformation. However, HUCB-MSCs from one of the 15 donors displayed an increased proliferation rate, failed to enter senescence, and exhibited an altered cell morphology. In this sample we observed two different cell phenotypes: one mesenchymal-like exhibited spindle shaped morphology and express specific mesenchymal surface markers (CD73, CD90, CD105, CD166) with low proliferation rate, and the second one with round, densely package epithelial-like cells with significantly increased proliferation rate. The PDT of epithelial-like populations was around 1day and 100% of cells were positive for proliferation marker Ki-67. Moreover, HUCB-MSCs showed a positive expression of human telomerase reverse transcriptase (hTERT), cMYC and exhibit increased number of CFU during the long-term culture in vitro. Furthermore, karyotype analysis revealed chromosomal abnormalities including duplications. Conclusions: Our studies demonstrate that HUCB-MSCs are susceptible to spontaneous malignant transformation during long-term culture. Spontaneous malignant transformation process following in vitro culture has enormous effect on the biosafety issues of future cell-based therapies and regenerative medicine regimens.

Keywords: mesenchymal stem cells, spontaneous, transformation, long-term culture

Procedia PDF Downloads 247

Authors: Tamer El-Messery, Teresa Sanchez-Moya, Ruben Lopez-Nicolas, Gaspar Ros, Esmat Aly

Abstract:

Olive leaves (OLs), the main by-product of the olive oil industry, have a considerable amount of phenolic compounds. The exploitation of these compounds represents the current trend in food processing. In this study, OLs polyphenols were microencapsulated with polycaprolactone (PCL) and utilized in formulating novel functional yoghurt. PCL-microcapsules were characterized by scanning electron microscopy, and Fourier transform infrared spectrometry analysis. Their total phenolic (TPC), total flavonoid (TFC) contents, and antioxidant activities (DPPH, FRAP, ABTS), and polyphenols bioaccessibility were measured after oral, gastric, and intestinal steps of in vitro digestion. The four yoghurt formulations (containing 0, 25, 50, and 75 mg of PCL-microsphere/100g yoghurt) were evaluated for their pH, acidity, syneresis viscosity, and color during storage. In vitro digestion significantly affected the phenolic composition in non-encapsulated extract while had a lower impact on encapsulated phenolics. Higher protection was provided for encapsulated OLs extract, and their higher release was observed at the intestinal phase. Yoghurt with PCL-microsphere had lower viscosity, syneresis, and color parameters, as compared to control yoghurt. Thus, OLs represent a valuable and cheap source of polyphenols which can be successfully applied, in microencapsulated form, to formulate functional yoghurt.

Keywords: yoghurt quality attributes, olive leaves, phenolic and flavonoids compounds, antioxidant activity, polycaprolactone as microencapsulant

Procedia PDF Downloads 125

Authors: Nina Tsvetkova, Rumen Harizanov, Aleksandra Ivanova, Iskra Rainova, Nina Yancheva-Petrova, Dimitar Strashimirov, Raina Enikova, Mihaela Videnova, Eleonora Kaneva, Iskren Kaftandjiev, Viktoria Levterova, Ivan Simeonovski, Nikolay Yanev, Georgi Hinkov

Abstract:

The Pneumocystis jirovecii (P. jirovecii) and protozoan of the genera Acanthamoeba, Cryptosporidium, and Toxoplasma gondii are opportunistic pathogens that can cause life-threatening infections in immunocompromised patients. Aim of the study was to evaluate the coinfection rate with opportunistic protozoal agents among Bulgarian patients diagnosed with P. jirovecii pneumonia. Thirty-eight pulmonary samples were collected from 38 patients (28 HIV-infected) with P. jirovecii infection. P. jirovecii DNA was detected by real-time PCR targeting the large mitochondrial subunit ribosomal RNA gene. Acanthamoeba was determined by genus-specific conventional PCR assay. Real-time PCR for the detection of a Toxoplasma gondii and Cryptosporidium DNA fragment was used. Pneumocystis DNA was detected in all 38 specimens; 28 (73.7%) were from HIV-infected patients. Three (10,7%) of them were co-infected with T. gondii and 1 (3.6%) with Cryptosporidium. In the group of non-HIV-infected (n=10), Cryptosporidium DNA was detected in an infant (10%). Acanthamoeba DNA was not found in the tested samples. The current study showed a relatively low rate of coinfections of Cryptosporidium spp./T. gondii and P. jirovecii in the Bulgarian patients studied.

Keywords: coinfection, opportunistic protozoal agents, Pneumocystis jirovecii, pulmonary infections

Procedia PDF Downloads 137

Authors: Vahid Changizi, Aram Rostami, Akbar Mosavi

Abstract:

Purpose of study: Critical macromolecules of cells such as DNA are in exposure to damage of free radicals that induced from interaction of ionizing radiation with biological systems. Selenium and vitamin-E are natural compound that has been shown to be a direct free radical scavenger. The aim of this study was to investigate the in vivo/in vitro radioprotective effect of selenium and vitamin-E separately and synergistically against genotoxicity induced by 6MV x-rays irradiation in cultured blood lymphocytes from 15 human volunteers. Methods: Fifteen volunteers were divided in three groups include A, B and C. These groups were given slenium(800 IU), vitamin-E(100 mg) and selenium(400 IU) + vitamin-E(50 mg), respectively. Peripheral blood samples were collected from each group before(0 hr) and 1, 2 and 3 hr after selenium and vitamin-E administration (separately and synergistically). Then the blood samples were irradiated to 200 cGy of 6 Mv x-rays. After that, lymphocyte samples were cultured with mitogenic stimulation to determine the chromosomal aberrations wih micronucleus assay in cytokinesis-blocked binucleated cells. Results: The lymphocytes in the blood samples collected at 1 hr after ingestion selenium and vitamin-E, exposed in vitro to x-rays exhibited a significant decrease in the incidence of micronuclei, compared with control group at 0 hr. The maximum protection and decrease in frequency of micronuclei(50%) was observed at 1 hr after administration of selenium and vitamin-E synergistically. Conclusion: The data suggest that ingestion of selenium and vitamin-E as a radioprotector substances before exposures may reduce genetic damage caused by x-rays irradiation.

Keywords: x-rays, selenium, vitamin-e, lymphocyte, micronuclei

Procedia PDF Downloads 258

Authors: Akbar Esmaeili, Zahra Salarieh

Abstract:

Cerium oxide nanoparticles (nano-series) are used as catalysts in industrial applications due to their free radical scavenging properties. Given that free radicals play an essential role in the pathology of many neurological diseases, we investigated the use of nanocrystals as a potential therapeutic agent for oxidative damage. This project synthesized nano-series from a new and environmentally friendly bio-pathway. Investigation of cerium nitrate in culture medium containing inoculated Lactobacillus acidophilus strain before incubation produces nano-series. Loaded with glatiramer acetate (GA) was formed by coating carboxymethylcellulose (CMC) and CeO2. FE-SEM analysis showed nano-series in the 9-11 nm range, spherical shape, and uniform particle size distribution. Cubic nanoparticles containing anti-multiple sclerosis (anti-Ms) treatment called GA were used. Glycerol monostearate (GMS) was used as a fat base, and evening primrose extract was used as an anti-inflammatory in cubosomes. Design-Expert® software was used to study the effects of different formulation factors on the properties of GAloaded cubic dispersions. Thirty GA-labeled cubic dispersions were prepared with GA-labeled carboxymethylcellulose and evaluated in vitro. The results showed an average nano-series size of 89.02 and a zeta potential of -49.9. Cubosomes containing GA-CMC/CeO2 showed a stable release profile for 180 min. The results showed that cubosomes containing GA-CMC/CeO2 could be a promising drug carrier with normal release behavior.

Keywords: ciochemistry, biotechnology, molecular, biology

Procedia PDF Downloads 26

Authors: Carlos Amnael Orozco-Diaz, Robert David Moorehead, Gwendolen Reilly, Fiona Gilchrist, Cheryl Ann Miller

Abstract:

The current gold-standard for maxillofacial reconstruction surgery (MRS) utilizes auto-grafted cancellous bone as a filler. This study was aimed towards developing a polylactic-acid/hydroxyapatite (PLA-HA) composite suitable for fused-deposition 3D printing. Functionalization of the polymer through the addition of HA was directed to promoting bone-regeneration properties so that the material can rival the performance of cancellous bone grafts in terms of bone-lesion repair. This kind of composite enables the production of MRS implants based off 3D-reconstructions from image studies – namely computed tomography – for anatomically-correct fitting. The present study encompassed in-vitro degradation and in-vitro biocompatibility profiling for 3D-printed PLA and PLA-HA composites. PLA filament (Verbatim Co.) and Captal S hydroxyapatite micro-scale HA powder (Plasma Biotal Ltd) were used to produce PLA-HA composites at 5, 10, and 20%-by-weight HA concentration. These were extruded into 3D-printing filament, and processed in a BFB-3000 3D-Printer (3D Systems Co.) into tensile specimens, and were mechanically challenged as per ASTM D638-03. Furthermore, tensile specimens were subjected to accelerated degradation in phosphate-buffered saline solution at 70°C for 23 days, as per ISO-10993-13-2010. This included monitoring of mass loss (through dry-weighing), crystallinity (through thermogravimetric analysis/differential thermal analysis), molecular weight (through gel-permeation chromatography), and tensile strength. In-vitro biocompatibility analysis included cell-viability and extracellular matrix deposition, which were performed both on flat surfaces and on 3D-constructs – both produced through 3D-printing. Discs of 1 cm in diameter and cubic 3D-meshes of 1 cm3 were 3D printed in PLA and PLA-HA composites (n = 6). The samples were seeded with 5000 MG-63 osteosarcoma-like cells, with cell viability extrapolated throughout 21 days via resazurin reduction assays. As evidence of osteogenicity, collagen and calcium deposition were indirectly estimated through Sirius Red staining and Alizarin Red staining respectively. Results have shown that 3D printed PLA loses structural integrity as early as the first day of accelerated degradation, which was significantly faster than the literature suggests. This was reflected in the loss of tensile strength down to untestable brittleness. During degradation, mass loss, molecular weight, and crystallinity behaved similarly to results found in similar studies for PLA. All composite versions and pure PLA were found to perform equivalent to tissue-culture plastic (TCP) in supporting the seeded-cell population. Significant differences (p = 0.05) were found on collagen deposition for higher HA concentrations, with composite samples performing better than pure PLA and TCP. Additionally, per-cell-calcium deposition on the 3D-meshes was significantly lower when comparing 3D-meshes to discs of the same material (p = 0.05). These results support the idea that 3D-printable PLA-HA composites are a viable resorbable material for artificial grafts for bone-regeneration. Degradation data suggests that 3D-printing of these materials – as opposed to other manufacturing methods – might result in faster resorption than currently-used PLA implants.

Keywords: bone regeneration implants, 3D-printing, in vitro testing, biocompatibility, polymer degradation, polymer-ceramic composites

Procedia PDF Downloads 146

Authors: Mike Wei, Nebu Koshy, Koen van Besien, Giorgio Inghirami, Steven M. Horwitz

Abstract:

T-cell prolymphocytic leukemia (T-PLL) is a rare hematologic disease characterized by a T-cell phenotype, rapid progression, and poor prognosis with median survival of less than a year. Alemtuzumab-based chemotherapy has increased the rate of complete remissions but these are often short-lived, and allogeneic transplant is considered the only curative therapy. In recent studies, JAK3 activating mutations have been identified in T-cell cancers, with T-PLL having the highest rate of JAK3 mutations (30 – 42%). As such, T-PLL is a model disease for evaluating the utility of JAK3 inhibitors. We present a case of a 64-year-old man with relapsed-refractory T-PLL. He was initially treated with alemtuzumab and obtained complete response and was consolidated with matched unrelated donor stem cell transplant. His disease stayed in remission for approximately 1.5 years before relapse, which was then treated with a clinical trial of romidepsin-lenalidomide (partial responses then progression at 6 months) and later alemtuzumab. Due to complications of myelosuppression and CMV reactivation, his treatment was interrupted leading to disease progression. The doubling time of lymphocyte count was approximately 20 days and over a span of 60 days the lymphocyte count rose from 8 x 109/L to 68 x 109/L. Exon sequencing showed a JAK3 mutation. The patient consented to and was treated with FDA-approved tofacitinib (initially 5 mg BID, increased to 10 mg BID after 15 days of treatment). An initial decrease in lymphocyte count was followed by progression. In vitro treatment of the patient’s cells showed modest effects of tofacitinib and ruxolitinib as single agents, in the range of doxorubicin, but synergy between the agents. After 40 days of treatment with tofacitinib and with a lymphocyte count of 150 x 109/L, ruxolitinib (5mg BID) was added. Over the 60 days since dual inhibition was started, the lymphocyte count has stabilized. The patient has remained completely asymptomatic during treatment with tofacitinib and ruxolitinib. Neutrophil count has remained normal. Platelet count and hemoglobin have however declined from ~50 x109/L to ~30 x109/L and from 11 g/dL to 8.1 g/dL respectively, since the introduction of ruxolitinib. The stabilization in lymphocyte count confirms the clinical activity of JAK inhibitors in T-PLL as suggested by the presence of JAK3 mutations and by in-vitro assays. It also suggests clinical synergy between ruxolitinib and tofacitinib in this setting. Prospective studies of JAK inhibitors in PLL patients with formal dose-finding studies are needed.

Keywords: tofacitinib, ruxolitinib, T-cell prolymphocytic leukemia, JAK3

Procedia PDF Downloads 296

Authors: Masoud Sakhinia, Sajjad Ahmad

Abstract:

Introduction: Though knowledge of the compositional makeup and structure of the limbal niche has progressed exponentially during the past decade, much is yet to be understood. Identifying the precise profile and role of the stromal makeup which spans the ocular surface may inform researchers of the most optimum conditions needed to effectively expand LESCs in vitro, whilst preserving their differentiation status and phenotype. Limbal fibroblasts, as opposed to corneal fibroblasts are thought to form an important component of the microenvironment where LESCs reside. Methods: The corneal stroma was tissue engineered in vitro using both limbal and corneal fibroblasts embedded within a tissue engineered 3D collagen matrix. The effect of these two different fibroblasts on LESCs and hTCEpi corneal epithelial cell line were then subsequently determined using phase contrast microscopy, histolological analysis and PCR for specific stem cell markers. The study aimed to develop an in vitro model which could be used to determine whether limbal, as opposed to corneal fibroblasts, maintained the stem cell phenotype of LESCs and hTCEpi cell line. Results: Tissue culture analysis was inconclusive and required further quantitative analysis for remarks on cell proliferation within the varying stroma. Histological analysis of the tissue-engineered cornea showed a comparable structure to that of the human cornea, though with limited epithelial stratification. PCR results for epithelial cell markers of cells cultured on limbal fibroblasts showed reduced expression of CK3, a negative marker for LESC’s, whilst also exhibiting a relatively low expression level of P63, a marker for undifferentiated LESCs. Conclusion: We have shown the potential for the construction of a tissue engineered human cornea using a 3D collagen matrix and described some preliminary results in the analysis of the effects of varying stroma consisting of limbal and corneal fibroblasts, respectively, on the proliferation of stem cell phenotype of primary LESCs and hTCEpi corneal epithelial cells. Although no definitive marker exists to conclusively illustrate the presence of LESCs, the combination of positive and negative stem cell markers in our study were inconclusive. Though it is less traslational to the human corneal model, the use of conditioned medium from that of limbal and corneal fibroblasts may provide a more simple avenue. Moreover, combinations of extracellular matrices could be used as a surrogate in these culture models.

Keywords: cornea, Limbal Stem Cells, tissue engineering, PCR

Procedia PDF Downloads 262

Authors: Priyanka Sharma, Niti Puri

Abstract:

Anemia develops when blood lacks enough healthy erythrocytes. Past studies indicated that anemia, inflammatory process, and oxidative stress are interconnected. Erythrocytes are continuously exposed to reactive oxygen species (ROS) during circulation, due to normal aerobic cellular metabolism and also pathology of inflammatory diseases. Systemic mastocytosis and genetic depletion of mast cells have been shown to affect anaemia. In the present study, we attempted to reveal whether mast cells have a direct role in clearance or erythrophagocytosis of normal or oxidatively damaged erythrocytes. Murine erythrocytes were treated with tert-butyl hydroperoxidase (t-BHP), an agent that induces oxidative damage and mimics in vivo oxidative stress. Normal and oxidatively damaged erythrocytes were labeled with carboxyfluorescein succinimidyl ester (CFSE) to track erythrophagocytosis. We show, for the first time, direct erythrophagocytosis of oxidatively damaged erythrocytes in vitro by RBL-2H3 mast cells as well as in vivo by murine peritoneal mast cells. Also, activated mast cells, as may be present in inflammatory conditions, showed a significant increase in the uptake of oxidatively damaged erythrocytes than resting mast cells. This suggests the involvement of mast cells in erythrocyte clearance during oxidative stress or inflammatory disorders. Partial inhibition of phagocytosis by various inhibitors indicated that this process may be controlled by several pathways. Hence, our study provides important evidence for involvement of mast cells in severe anemia due to inflammation and oxidative stress and might be helpful to circumvent the adverse anemic disorders.

Keywords: mast cells, anemia, erythrophagocytosis, oxidatively damaged erythrocytes

Procedia PDF Downloads 238