In vitro studies of nanosilver-doped titanium implants for oral and maxillofacial surgery PubMed Central Pokrowiecki, RafaÅ; ZarÄba, Tomasz; Szaraniec, Barbara; PaÅka, Krzysztof; Mielczarek, Agnieszka; Menaszek, ElÅ¼bieta; Tyski, Stefan 2017-01-01 The addition of an antibacterial agent to dental implants may provide the opportunity to decrease the percentage of implant failures due to peri-implantitis. For this purpose, in this study, the potential efficacy of nanosilver-doped titanium biomaterials was determined. Titanium disks were incorporated with silver nanoparticles over different time periods by Tollens reaction, which is considered to be an eco-friendly, cheap, and easy-to-perform method. The surface roughness, wettability, and silver release profile of each disc were measured. In addition, the antibacterial activity was also evaluated by using disk diffusion tests for bacteria frequently isolated from the peri-implant biofilm: Streptococcus mutans, Streptococcus mitis, Streptococcus oralis, Streptococcus sanguis, Porphyromonas gingivalis, Staphylococcus aureus, and Escherichia coli. Cytotoxicity was evaluated in vitro in a natural human osteoblasts cell culture. The addition of nanosilver significantly increased the surface roughness and decreased the wettability in a dose-dependent manner. These surfaces were significantly toxic to all the tested bacteria following a 48-hour exposure, regardless of silver doping duration. A concentration of 0.05 ppm was sufficient to inhibit Gram-positive and Gram-negative species, with the latter being significantly more susceptible to silver ions. However, after the exposure of human osteoblasts to 0.1 ppm of silver ions, a significant decrease in cell viability was observed by using ToxiLightâ¢ BioAssay Kit after 72 hours. Data from the present study indicated that the incorporation of nanosilver may influence the surface properties that are important in the implant healing process. The presence of nanosilver on the titanium provides an antibacterial activity related to the bacteria involved in peri-implantitis. Finally, the potential toxicological considerations of nanosilver should further be investigated, as both the antibacterial and cytotoxic properties

A Novel In Vitro Model for Studying Quiescence and Activation of Primary Isolated Human Myoblasts PubMed Central Sellathurai, Jeeva; Cheedipudi, Sirisha; Dhawan, Jyotsna; SchrÃ¸der, Henrik Daa 2013-01-01 Skeletal muscle stem cells, satellite cells, are normally quiescent but become activated upon muscle injury. Recruitment of resident satellite cells may be a useful strategy for treatment of muscle disorders, but little is known about gene expression in quiescent human satellite cells or the mechanisms involved in their early activation. We have developed a method to induce quiescence in purified primary human myoblasts isolated from healthy individuals. Analysis of the resting state showed absence of BrdU incorporation and lack of KI67 expression, as well as the extended kinetics during synchronous reactivation into the cell cycle, confirming arrest in the G0 phase. Reactivation studies showed that the majority (>95%) of the G0 arrested cells were able to re-enter the cell cycle, confirming reversibility of arrest. Furthermore, a panel of important myogenic factors showed expression patterns similar to those reported for mouse satellite cells in G0, reactivated and differentiated cultures, supporting the applicability of the human model. In addition, gene expression profiling showed that a large number of genes (4598) were differentially expressed in cells activated from G0 compared to long term exponentially proliferating cultures normally used for in vitro studies. Human myoblasts cultured through many passages inevitably consist of a mixture of proliferating and non-proliferating cells, while cells activated from G0 are in a synchronously proliferating phase, and therefore may be a better model for in vivo proliferating satellite cells. Furthermore, the temporal propagation of proliferation in these synchronized cultures resembles the pattern seen in vivo during regeneration. We therefore present this culture model as a useful and novel condition for molecular analysis of quiescence and reactivation of human myoblasts. PMID:23717533

[Fracture resistance of Procera Allceram depending on the framework design--an in vitro study]. PubMed Hagmann, Edgar; Marinello, Carlo P; Zitzmann, Nicola U 2006-01-01 Procera AllCeram is one of the all-ceramic systems with an aluminium-oxide core employing CAD/CAM technology. The aim of the current study was to investigate the fracture resistance of Procera AllCeram full-ceramic crowns with a reduced core design compared to the conventional method. In addition, a possible influence of the preparation form (molars or premolars) and the cementation material (glas-ionomer or composite) was analyzed. For both preparation forms, 30 ceramic cores with reduced margins (collarless cores, test) and 30 cores with extended cores (control) were veneered with porcelain in a standardized procedure (total 120 crowns). For the test group, Procera-AllCeram-margin ceramic material was used for the porcelain collar. 40 crowns each were cemented on stainless steel dies with either Ketac-Cem Aplicap or Panavia F. The additional 40 crowns were set on polyurethane dies without cementation and occlusally loaded until fracture occurred. Among the molar crowns, no differences were observed in fracture resistance neither for the different core designs (test or control) nor for the cementation materials. For the premolar form, fusing of a porcelain margin was associated with a reduction in fracture resistance, while the use of composite cement was accompanied with an increase. The present in vitro results indicate that for Procera AllCeram crowns with a highly undulating preparation margin, a conventional core design combined with adhesive cementation is preferable, especially in the posterior region due to higher chewing forces; this assumption needs to be proven in clinical studies.

Comparative assessment of antimicrobial efficacy of different hand sanitizers: An in vitro study PubMed Central Jain, Vardhaman Mulchand; Karibasappa, Gundabaktha Nagappa; Dodamani, Arun Suresh; Prashanth, Vishwakarma K.; Mali, Gaurao Vasant 2016-01-01 Background: To evaluate the antimicrobial efficacy of four different hand sanitizers against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis as well as to assess and compare the antimicrobial effectiveness among four different hand sanitizers. Materials and Methods: The present study is an in vitro study to evaluate antimicrobial efficacy of Dettol, Lifebuoy, PureHands, and Sterillium hand sanitizers against clinical isolates of the aforementioned test organisms. The well variant of agar disk diffusion test using Mueller-Hinton agar was used for evaluating the antimicrobial efficacy of hand sanitizers. McFarland 0.5 turbidity standard was taken as reference to adjust the turbidity of bacterial suspensions. Fifty microliters of the hand sanitizer was introduced into each of the 4 wells while the 5th well incorporated with sterile water served as a control. This was done for all the test organisms and plates were incubated in an incubator for 24 h at 37ÎC. After incubation, antimicrobial effectiveness was determined using digital caliper (mm) by measuring the zone of inhibition. Results: The mean diameters of zones of inhibition (in mm) observed in Group A (Sterillium), Group B (PureHands), Group C (Lifebuoy), and Group D (Dettol) were 22 Â± 6, 7.5 Â± 0.5, 9.5 Â± 1.5, and 8 Â± 1, respectively. Maximum inhibition was found with Group A against all the tested organisms. Data were statistically analyzed using analysis of variance, followed by post hoc test for group-wise comparisons. The difference in the values of different sanitizers was statistically significant at P < 0.001. Conclusion: Sterillium was the most effective hand sanitizer to maintain the hand hygiene. PMID:27857768

A miniaturized glucose biosensor for in vitro and in vivo studies. PubMed Yang, Yang-Li; Huang, Jian-Feng; Tseng, Ta-Feng; Lin, Chia-Ching; Lou, Shyh-Liang 2008-01-01 A miniaturized wireless glucose biosensor has been developed to perform in vitro and in vivo studies. It consists of an external control subsystem and an implant sensing subsystem. The implant subsystem consists of a micro-processor, which coordinates circuitries of radio frequency, power regulator, command demodulator, glucose sensing trigger and signal read-out. Except for a set of sensing electrodes, the micro-processor, the circuitries and a receiving coil were hermetically sealed with polydimethylsiloxane. The electrode set is a substrate of silicon oxide coated with platinum, which includes a working electrode and a reference electrode. Glucose oxidase was immobilized on the surface of the working electrode. The implant subsystem bi-directionally communicates with the external subsystem via radio frequency technologies. The external subsystem wirelessly supplies electricity to power the implant, issues commands to the implant to perform tasks, receives the glucose responses detected by the electrode, and relays the response signals to a computer through a RS-232 connection. Studies of in vitro and in vivo were performed to evaluate the biosensor. The linear response of the biosensor is up to 15 mM of glucose in vitro. The results of in vivo study show significant glucose variations measured from the interstitial tissue fluid of a diabetes rat in fasting and non-fasting periods.

Pasteurization of bone for tumour eradication prior to reimplantation â An in vitro & pre-clinical efficacy study PubMed Central Kode, Jyoti; Taur, Prasad; Gulia, Ashish; Jambhekar, Nirmala; Agarwal, Manish; Puri, Ajay 2014-01-01 Background & objectives: In current era of limb-salvage therapy, pasteurization of bone sarcomas is receiving growing attention as a potential extracorporeal treatment and cost-effective alternative to allografts and radiation before surgical reimplantation. Detailed in vitro and in vivo pre-clinical study to evaluate efficacy of pasteurization to eradicate malignant cells has not been reported yet. The present study was carried out to assess the efficacy of pasteurization to kill tumour cells both in vitro and in vivo. Methods: Surgically resected specimens of osteosarcomas (n=4) were cut into equal halves and one section was pasteurized by heating at 60Â°C to 65Â°C for 40 min. Paired samples before and after pasteurization were studied in vitro for DNA ploidy, evaluation of histological change and elimination of mitotic activity. These tissues were transplanted in immune-deficient NOD-SCID mice to evaluate effect on tumour-generating ability, presence of human nuclei, osteopontin and cytokine/chemokines released in tumour-transplanted mice. Results: Non-pasteurized tumour samples had viable tumour cells which exhibited significant growth in culture, increased proliferative ability and clonogenic potential while respective pasteurized tumour tissues did not grow in culture and did not exhibit clonogenicity. Flow cytometry revealed that propidium iodide positive dead cells increased significantly (P< 0.01) post pasteurization. Seven of 12 non-pasteurized tumour transplanted mice demonstrated tumour-forming ability as against 0 of 12 in pasteurized tumour transplanted mice. Solid tumour xenografts exhibited strong expression of anti-human nuclei and osteopontin by immunohistochemistry as well as secretary human interluekin-6 (IL-6) while pasteurized mice failed to express these markers. Interpretation & conclusions: This study has provided a basis to establish pasteurization as being efficacious in ensuring tumour eradication from resected bone tumour specimens

Technical Advance: New in vitro method for assaying the migration of primary B cells using an endothelial monolayer as substrate. PubMed Stewart-Hutchinson, Phillip J; Szasz, Taylor P; Jaeger, Emily R; Onken, Michael D; Cooper, John A; Morley, Sharon Celeste 2017-09-01 Migration of B cells supports their development and recruitment into functional niches. Therefore, defining factors that control B cell migration will lead to a better understanding of adaptive immunity. In vitro cell migration assays with B cells have been limited by poor adhesion of cells to glass coated with adhesion molecules. We have developed a technique using monolayers of endothelial cells as the substrate for B cell migration and used this technique to establish a robust in vitro assay for B cell migration. We use TNF-Î± to up-regulate surface expression of the adhesion molecule VCAM-1 on endothelial cells. The ligand VLA-4 is expressed on B cells, allowing them to interact with the endothelial monolayer and migrate on its surface. We tested our new method by examining the role of L-plastin (LPL), an F-actin-bundling protein, in B cell migration. LPL-deficient (LPL -/- ) B cells displayed decreased speed and increased arrest coefficient compared with wild-type (WT) B cells, following chemokine stimulation. However, the confinement ratios for WT and LPL -/- B cells were similar. Thus, we demonstrate how the use of endothelial monolayers as a substrate will support future interrogation of molecular pathways essential to B cell migration. Â© Society for Leukocyte Biology.

Evaluation of an in vitro method for acaricidal effect. Activity of parathion, phosmet and phoxim against Sarcoptes scabiei. PubMed Brimer, L; Henriksen, S A; Gyrd-Hansen, N; Rasmussen, F 1993-12-01 An in vitro test to determine the acaricidal effect of organophosphorous insecticides (OP) is described. The effect of parathion, phoxim and phosmet against the pig mange mite Sarcoptes scabiei var. suis was evaluated. The test is based on the migration ability of mites on the surface of agar gels containing the acaricide. The mite activity is expressed as a migration index (MI) and compared with the OP concentration in the agar. Good dose-response data were obtained for all three OPs tested, although the instability of phosmet required special precautions concerning the analysis of the agar. The test was found to be accurate, sensitive, easy to carry out and applicable for routine determinations. However, the test requires that the actual concentrations of the OPs in the gel batches are determined. For the three OPs used analytical methods were developed. While the lower threshold for acaricidal effect in vitro was approximately 1-2 micrograms g-1 for all three OPs tested, a significant difference in the higher concentration range was seen between the dose-response curve for parathion and the curves for phoxim and phosmet. While the latter curves decreased only slightly at concentrations above 3-6 micrograms g-1 (corresponding to MI values around 5-10), the curve for parathion was linear down to an MI value of 1, corresponding to a parathion concentration of approximately 30 micrograms g-1. This discrepancy was ascribed to different rates of uptake through the cuticula due to differences in the lipophilicity of the OPs.

In Vitro, In Vivo and Post Explantation Testing of Glucose-Detecting Biosensors: Current Methods and Recommendations PubMed Central Koschwanez, Heidi E.; Reichert, W. Monty 2007-01-01 To date, there have been a number of cases where glucose sensors have performed well over long periods of implantation; however, it remains difficult to predict whether a given sensor will perform reliably, will exhibit gradual degradation of performance, or will fail outright soon after implantation. Typically, the literature emphasizes the sensor that performed well, while only briefly (if at all) mentioning the failed devices. This leaves open the question of whether current sensor designs are adequate for the hostile in vivo environment, and whether these sensors have been assessed by the proper regimen of testing protocols. This paper reviews the current in vitro and in vivo testing procedures used to evaluate the functionality and biocompatibility of implantable glucose sensors. An overview of the standards and regulatory bodies that govern biomaterials and end-product device testing precedes a discussion of up-to-date invasive and non-invasive technologies for diabetes management. Analysis of current in vitro, in vivo, and then post implantation testing is presented. Given the underlying assumption that the success of the sensor in vivo foreshadows the long-term reliability of the sensor in the human body, the relative merits of these testing methods are evaluated with respect to how representative they are of human models. PMID:17524479

SkinEthic Laboratories, a company devoted to develop and produce in vitro alternative methods to animal use. PubMed de Brugerolle, Anne 2007-01-01 SkinEthic Laboratories is a France-based biotechnology company recognised as the world leader in tissue engineering. SkinEthic is devoted to develop and produce reliable and robust in vitro alternative methods to animal use in cosmetic, chemical and pharmaceutical industries. SkinEthic models provide relevant tools for efficacy and safety screening tests in order to support an integrated decision-making during research and development phases. Some screening tests are referenced and validated as alternatives to animal use (Episkin), others are in the process of validation under ECVAM and OECD guidelines. SkinEthic laboratories provide a unique and joined experience of more than 20 years from Episkin SNC and SkinEthic SA. Their unique cell culture process allows in vitro reconstructed human tissues with well characterized histology, functionality and ultrastructure features to be mass produced. Our product line includes skin models: a reconstructed human epidermis with a collagen layer, Episkin, reconstructed human epidermis without or with melanocytes (with a tanning degree from phototype II to VI) and a reconstructed human epithelium, i.e. cornea, and other mucosa, i.e. oral, gingival, oesophageal and vaginal. Our philosophy is based on 3 main commitments: to support our customers by providing robust and reliable models, to ensure training and education in using validated protocols, allowing a large array of raw materials, active ingredients and finished products in solid, liquid, powder, cream or gel form to be screened, and, to provide a dedicated service to our partners.

Comparison of manual and homogenizer methods for preparation of tick-derived stabilates of Theileria parva: equivalence testing using an in vitro titration model. PubMed Mbao, V; Speybroeck, N; Berkvens, D; Dolan, T; Dorny, P; Madder, M; Mulumba, M; Duchateau, L; Brandt, J; Marcotty, T 2005-07-01 Theileria parva sporozoite stabilates are used in the infection and treatment method of immunization, a widely accepted control option for East Coast fever in cattle. T. parva sporozoites are extracted from infected adult Rhipicephalus appendiculatus ticks either manually, using a pestle and a mortar, or by use of an electric homogenizer. A comparison of the two methods as a function of stabilate infectivity has never been documented. This study was designed to provide a quantitative comparison of stabilates produced by the two methods. The approach was to prepare batches of stabilate by both methods and then subject them to in vitro titration. Equivalence testing was then performed on the average effective doses (ED). The ratio of infective sporozoites yielded by the two methods was found to be 1.14 in favour of the manually ground stabilate with an upper limit of the 95% confidence interval equal to 1.3. We conclude that the choice of method rests more on costs, available infrastructure and standardization than on which method produces a richer sporozoite stabilate.

In vitro cell and tissue models for studying host-microbe interactions: a review. PubMed Bermudez-Brito, Miriam; Plaza-DÃ­az, Julio; Fontana, Luis; MuÃ±oz-Quezada, Sergio; Gil, Angel 2013-01-01 Ideally, cell models should resemble the in vivo conditions; however, in most in vitro experimental models, epithelial cells are cultivated as monolayers, in which the establishment of functional epithelial features is not achieved. To overcome this problem, co-culture experiments with probiotics, dendritic cells and intestinal epithelial cells and three-dimensional models attempt to reconcile the complex and dynamic interactions that exist in vivo between the intestinal epithelium and bacteria on the luminal side and between the epithelium and the underlying immune system on the basolateral side. Additional models include tissue explants, bioreactors and organoids. The present review details the in vitro models used to study host-microbe interactions and explores the new tools that may help in understanding the molecular mechanisms of these interactions.

Combining microfluidics, optogenetics and calcium imaging to study neuronal communication in vitro. PubMed Renault, Renaud; Sukenik, Nirit; Descroix, StÃ©phanie; Malaquin, Laurent; Viovy, Jean-Louis; Peyrin, Jean-Michel; Bottani, Samuel; Monceau, Pascal; Moses, Elisha; Vignes, MaÃ©va 2015-01-01 In this paper we report the combination of microfluidics, optogenetics and calcium imaging as a cheap and convenient platform to study synaptic communication between neuronal populations in vitro. We first show that Calcium Orange indicator is compatible in vitro with a commonly used Channelrhodopsine-2 (ChR2) variant, as standard calcium imaging conditions did not alter significantly the activity of transduced cultures of rodent primary neurons. A fast, robust and scalable process for micro-chip fabrication was developed in parallel to build micro-compartmented cultures. Coupling optical fibers to each micro-compartment allowed for the independent control of ChR2 activation in the different populations without crosstalk. By analyzing the post-stimuli activity across the different populations, we finally show how this platform can be used to evaluate quantitatively the effective connectivity between connected neuronal populations.

Vasorelaxant effect of quercetin on cerebral basilar artery in vitro and the underlying mechanisms study. PubMed Yuan, Tian-Yi; Niu, Zi-Ran; Chen, Di; Chen, Yu-Cai; Zhang, Hui-Fang; Fang, Lian-Hua; Du, Guan-Hua 2018-04-25 The aim of this study is to investigate the vasorelaxant effect of quercetin on cerebral basilar artery in vitro and provide a preliminary discussion concerning the underlying mechanisms. Using a DMT-isolated micro vessel system, quercetin was found to exhibit a vasodilatory effect on basilar arteries contracted by potassium chloride (KCl), endothelin-1 (ET-1), and 5-hydroxytryptamine (5-HT). The vasorelaxant effect of quercetin was partially attenuated when endothelium cells were removed. L-NAME, indomethacin, and ODQ treatment also decreased the potency of quercetin. In endothelium-denuded rings, the vasorelaxant effect of quercetin was not influenced by K + channel inhibitors. However, quercetin inhibited KCl induced extracellular calcium influx and ET-1 induced transient intracellular calcium release in a Ca 2+ -free solution. In conclusion, quercetin induced relaxation of the basilar artery in vitro is partially dependent on endothelium, which is mainly related to NO and COX pathways. It also induces relaxation through blockage of calcium channels.

In vitro terahertz spectroscopy of gelatin-embedded human brain tumors: a pilot study NASA Astrophysics Data System (ADS) Chernomyrdin, N. V.; Gavdush, A. A.; Beshplav, S.-I. T.; Malakhov, K. M.; Kucheryavenko, A. S.; Katyba, G. M.; Dolganova, I. N.; Goryaynov, S. A.; Karasik, V. E.; Spektor, I. E.; Kurlov, V. N.; Yurchenko, S. O.; Komandin, G. A.; Potapov, A. A.; Tuchin, V. V.; Zaytsev, K. I. 2018-04-01 We have performed the in vitro terahertz (THz) spectroscopy of human brain tumors. In order to fix tissues for the THz measurements, we have applied the gelatin embedding. It allows for preserving tissues from hydration/dehydration and sustaining their THz response similar to that of the freshly-excised tissues for a long time after resection. We have assembled an experimental setup for the reflection-mode measurements of human brain tissues based on the THz pulsed spectrometer. We have used this setup to study in vitro the refractive index and the amplitude absorption coefficient of 2 samples of malignant glioma (grade IV), 1 sample of meningioma (grade I), and samples of intact tissues. We have observed significant differences between the THz responses of normal and pathological tissues of the brain. The results of this paper highlight the potential of the THz technology in the intraoperative neurodiagnosis of tumors relying on the endogenous labels of tumorous tissues.

Black pepper constituent piperine: genotoxicity studies in vitro and in vivo. PubMed Thiel, Anette; Buskens, Carin; Woehrle, Tina; Etheve, StÃ©phane; Schoenmakers, Ankie; Fehr, Markus; Beilstein, Paul 2014-04-01 Piperine is responsible for the hot taste of black pepper. Publications on genotoxicity of piperine are reported: negative Ames Tests and one in vitro micronucleus test (MNT). In vivo tests were mainly negative. In the majority of the data the administered dose levels did not follow the dose selection requirements of regulatory guidelines of having dose levels up to the maximum tolerated dose (MTD). The only oral high dose studies were a positive in vivo MNT in mice in contrast to a negative in vivo chromosome aberration test in rats. Thus, conflicting results in genotoxicity testing are published. To investigate this further, we administered piperine to mice up to the MTD and determined micronuclei-frequency. Piperine reduces core body temperature and interferes with blood cells both being known to result in irrelevant positive in vivo MNTs. Therefore we added mechanistic endpoints: core body temperature, haematology, erythropoietin level, and organ weights. Additionally an in vitro MNT in Chinese hamster ovary cells was performed. Piperine was negative in the in vitro MNT. It caused significant reduction of core body temperature, decrease of white blood cells and spleen weights but no increase in the micronucleus-frequency. Thus, in our studies piperine was not genotoxic. Copyright Â© 2014 Elsevier Ltd. All rights reserved.

A new alternative method for testing skin irritation using a human skin model: a pilot study. PubMed Miles, A; Berthet, A; Hopf, N B; Gilliet, M; Raffoul, W; Vernez, D; Spring, P 2014-03-01 Studies assessing skin irritation to chemicals have traditionally used laboratory animals; however, such methods are questionable regarding their relevance for humans. New in vitro methods have been validated, such as the reconstructed human epidermis (RHE) model (EpiskinÂ®, EpidermÂ®). The comparison (accuracy) with in vivo results such as the 4-h human patch test (HPT) is 76% at best (EpidermÂ®). There is a need to develop an in vitro method that better simulates the anatomo-pathological changes encountered in vivo. To develop an in vitro method to determine skin irritation using human viable skin through histopathology, and compare the results of 4 tested substances to the main in vitro methods and in vivo animal method (Draize test). Human skin removed during surgery was dermatomed and mounted on an in vitro flow-through diffusion cell system. Ten chemicals with known non-irritant (heptylbutyrate, hexylsalicylate, butylmethacrylate, isoproturon, bentazon, DEHP and methylisothiazolinone (MI)) and irritant properties (folpet, 1-bromohexane and methylchloroisothiazolinone (MCI/MI)), a negative control (sodiumchloride) and a positive control (sodiumlaurylsulphate) were applied. The skin was exposed at least for 4h. Histopathology was performed to investigate irritation signs (spongiosis, necrosis, vacuolization). We obtained 100% accuracy with the HPT model; 75% with the RHE models and 50% with the Draize test for 4 tested substances. The coefficients of variation (CV) between our three test batches were <0.1, showing good reproducibility. Furthermore, we reported objectively histopathological irritation signs (irritation scale): strong (folpet), significant (1-bromohexane), slight (MCI/MI at 750/250ppm) and none (isoproturon, bentazon, DEHP and MI). This new in vitro test method presented effective results for the tested chemicals. It should be further validated using a greater number of substances; and tested in different laboratories in order to suitably

Methods for the isolation, culture and assessment of the status of anaerobic rumen chytrids in both in vitro and in vivo systems. PubMed Rezaeian, Mohammad; Beakes, Gordon W; Parker, David S 2004-10-01 Anaerobic fungi were isolated from both the rumen and faeces of nine sheep and a cow. A reliable and simple method for the isolation of anaerobic fungi using 24 h rumen incubated milled straw as the inoculum source was developed. We also evaluate the use of chitin measurements as an assay of rumen fungal biomass. Chitin levels were determined from various sample sources (milled barley straw used as the fungal culture substrate in vitro; plant particulate digests from the rumen (PLP) and centrifuged strained rumen fluid (CSRF) using both HPLC and colorimetric methods. Both methods were highly correlated and consequently the simpler colorimetric method was adopted for subsequent studies. There was also a high degree of correlation between anaerobic fungal cellulase activities with the assayed chitin content of milled barley straw cultures over 12 d of an in vitro experiment. The colorimetric chitin assay protocol was then used to assess the diurnal variation and abundance of rumen fungi in in vivo assays. We assessed the distribution of chitin (mg g(-1) dry matter) in various fractions of the strained rumen fluid (SRF) and PLP samples from the rumen of sheep. Chitin was detected in all fractions of strained rumen fluid but the main source of chitin in the samples may be attributed to the fungal biomass. We did not detect any significant differences in chitin levels over a 24 h sampling period. Finally, an SEM study on subsamples of milled straw and plant particulate matter used in the chitin assays, revealed that the pattern of the fungal development on substrate material differs from the culture medium to the rumen.

Antioxidants, endothelial dysfunction, and DCS: in vitro and in vivo study. PubMed Wang, Qiong; Mazur, Aleksandra; Guerrero, FranÃ§ois; Lambrechts, Kate; Buzzacott, Peter; Belhomme, Marc; Theron, MichaÃ«l 2015-12-15 Reactive oxygen species (ROS) production is a well-known effect in individuals after an undersea dive. This study aimed to delineate the links between ROS, endothelial dysfunction, and decompression sickness (DCS) through the use of antioxidants in vitro and in vivo. The effect of N-acetylcysteine (NAC) on superoxide and peroxynitrite, nitric oxide (NO) generation, and cell viability during in vitro diving simulation were analyzed. Also analyzed was the effect of vitamin C and NAC on plasma glutathione thiol and thiobarbituric acid reactive substances (TBARS), plasma angiotensin-converting enzyme (ACE) activity, and angiotensin-II and DCS morbidity during in vivo diving simulation. During an in vitro diving simulation, vascular endothelial cells showed overproduction of superoxide and peroxynitrite, obvious attenuation of NO generation, and promotion of cell death, all of which were reversed by NAC treatment. After in vivo diving simulation, plasma ACE activity and angiotensin-II level were not affected. The plasma level of glutathione thiol was downregulated after the dive, which was attenuated partially by NAC treatment. Plasma TBARS level was upregulated; however, either NAC or vitamin C treatment failed to prevent DCS morbidity. During in vitro simulation, endothelial superoxide and peroxynitrite-mediated oxidative stress were involved in the attenuation of NO availability and cell death. This study is the first attempt to link oxidative stress and DCS occurrence, and the link could not be confirmed in vivo. Even in the presence of antioxidants, ROS and bubbles generated during diving and/or decompression might lead to embolic or biochemical stress and DCS. Diving-induced oxidative stress might not be the only trigger of DCS morbidity. Copyright Â© 2015 the American Physiological Society.