An Efficient Protocol For The Generation Of Monocyte Derived Dendritic Cells Using Serum-free Media For Clinical Applications In Post Remission Aml Patients.

Protocols for the generation of dendritic cells (DCs) using serum as a supplementation of culture media leads to reactions due to animal proteins and disease transmissions. Several types of serum-free media (SFM), based on good manufacture practices (GMP), have recently been used and seem to be a viable option. The aim of this study was to evaluate the results of the differentiation, maturation, and function of DCs from Acute Myeloid Leukemia patients (AML), generated in SFM and medium supplemented with autologous serum (AS). DCs were analyzed by phenotype characteristics, viability, and functionality. The results showed the possibility of generating viable DCs in all the conditions tested. In patients, the X-VIVO 15 medium was more efficient than the other media tested in the generation of DCs producing IL-12p70 (p=0.05). Moreover, the presence of AS led to a significant increase of IL-10 by DCs as compared with CellGro (p=0.05) and X-Vivo15 (p=0.05) media, both in patients and donors. We concluded that SFM was efficient in the production of DCs for immunotherapy in AML patients. However, the use of AS appears to interfere with the functional capacity of the generated DCs.

Growth Factor Stimulation Improves The Structure And Properties Of Scaffold-free Engineered Auricular Cartilage Constructs.

The reconstruction of the external ear to correct congenital deformities or repair following trauma remains a significant challenge in reconstructive surgery. Previously, we have developed a novel approach to create scaffold-free, tissue engineering elastic cartilage constructs directly from a small population of donor cells. Although the developed constructs appeared to adopt the structural appearance of native auricular cartilage, the constructs displayed limited expression and poor localization of elastin. In the present study, the effect of growth factor supplementation (insulin, IGF-1, or TGF-β1) was investigated to stimulate elastogenesis as well as to improve overall tissue formation. Using rabbit auricular chondrocytes, bioreactor-cultivated constructs supplemented with either insulin or IGF-1 displayed increased deposition of cartilaginous ECM, improved mechanical properties, and thicknesses comparable to native auricular cartilage after 4 weeks of growth. Similarly, growth factor supplementation resulted in increased expression and improved localization of elastin, primarily restricted within the cartilaginous region of the tissue construct. Additional studies were conducted to determine whether scaffold-free engineered auricular cartilage constructs could be developed in the 3D shape of the external ear. Isolated auricular chondrocytes were grown in rapid-prototyped tissue culture molds with additional insulin or IGF-1 supplementation during bioreactor cultivation. Using this approach, the developed tissue constructs were flexible and had a 3D shape in very good agreement to the culture mold (average error <400 µm). While scaffold-free, engineered auricular cartilage constructs can be created with both the appropriate tissue structure and 3D shape of the external ear, future studies will be aimed assessing potential changes in construct shape and properties after subcutaneous implantation.

The People's Stage in Imperial Germany: Social Democracy and Culture 1890-1914

The clash between German Social Democracy--the party, intellectuals and workers--and the German Imperial State was played out in the Freie Volksbahne (Free People's Theatre) founded by intellectuals to energise working class political awareness of drama with a political and social cutting edge. It fell foul of state censorship, lost its bite, yet prospered.

Purification of recombinant human growth hormone from CHO cell culture supernatant by Gradiflow preparative electrophoresis technology

Purification of recombinant human growth hormone (rhGH) from Chinese hamster ovary (CHO) cell culture supernatant by Gradiflow large-scale electrophoresis is described. Production of rhGH in CHO cells is an alternative to production in Escherichia coli, with the advantage that rhGH is secreted into protein-free production media, facilitating a more simple purification and avoiding resolubilization of inclusion bodies and protein refolding. As an alternative to conventional chromatography, rhGH was purified in a one-step procedure using Gradiflow technology. Clarified culture supernatant containing rhGH was passed through a Gradiflow BF200 and separations were performed over 60 min using three different buffers of varying pH. Using a 50 mM Tris/Hepes buffer at pH 7.5 together with a 50 kDa separation membrane, rhGH was purified to approximately 98% purity with a yield of 90%. This study demonstrates the ability of Gradiflow preparative electrophoresis technology to purify rhGH from mammalian cell culture supernatant in a one-step process with high purity and yield. As the Gradiflow is directly scalable, this study also illustrates the potential for the inclusion of the Gradiflow into bioprocesses for the production of clinical grade rhGH and other therapeutic proteins. (C) 2003 Elsevier Science (USA). All rights reserved.

Formation Of The Maritime Labor Force In Brazil: Culture And Daily Life, Tradition And Resistance (1808-1850).

Formation Of The Maritime Labor Force In Brazil: Culture And Daily Life, Tradition And Resistance (1808-1850). Since the 16(th) Century, Brazil has played a major role in the rise of a new economical and social order, in which ships represented a space of struggle and contradictions among rulers, captains and sailors. This article will study the proletarization process that transformed Indians, small farmers, free and slave black people in maritime labor force in Brazil during the first half of 19(th) century.

The kinetics of baculovirus adsorption to insect cells in suspension culture

The influence of various culture parameters on the attachment of a recombinant baculovirus to suspended insect cells was examined under normal culture conditions. These parameters included cell density, multiplicity of infection, and composition of the cell growth medium. It was found that the fractional rate of virus attachment was independent of the multiplicity of infection but dependent on the cell density. A first order mathematical model was used to simulate the adsorption kinetics and predict the efficiency of virus attachment under the various culture conditions. This calculated efficiency of virus attachment was observed to decrease at high cell densities, which was attributed to cell clumping. It was also observed that virus attachment was more efficient in Sf900II serum free medium than it was in IPL-41 serum-supplemented medium. This effect was attributed to the protein in serum which may coat the cells and so inhibit adsorption. A general discussion relating the observations made in-these experiments to the kinetics of recombinant baculovirus adsorption to suspended insect cells is presented.

Chinese hamster ovary cells produce sufficient recombinant insulin-like growth factor I to support growth in serum-free medium - Serum-free growth of IGF-I producing CHO cells

Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture, and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/10(6) cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin.

Adipose tissue mesenchymal stem cell expansion in animal serum-free medium supplemented with autologous human platelet lysate

BACKGROUND: Mesenchymal stem cells (MSCs) have been considered for human regenerative therapy applications, and safe culture and expansion protocols are needed especially in the context of interspecies contamination. Human platelet lysate (PL) has been proposed as animal serum substitute during in vitro MSC expansion. In this work, a simplified and efficient method to obtain autologous PL to replace animal serum in cell culture applications is described. STUDY DESIGN AND METHODS: PL obtained by freezing and centrifugation procedures was tested as medium supplement for human adipose mesenchymal stem cell (hASC) culture. Differential proliferation, immunophenotypic changes, and differentiation under PL or fetal bovine serum (FBS) were assessed. RESULTS: In contrast to 10% FBS supplementation, cell population doubling time was significantly lower when hASCs were cultured with the same concentration of PL ( PL 22.9 +/- 1.5 hr vs. FBS 106.7 +/- 6.5 hr, t test, p < 0.05). Furthermore, hASCs maintained with 2.5% PL supplementation also showed satisfactory results. Immunophenotypic analysis revealed no differences between hASCs cultivated with PL or FBS supplementation and both cultures retained the potential to differentiate into adipose cells. These results demonstrate that autologous PL obtained from the same donor can be used as animal serum substitute in hASC culture. CONCLUSIONS: Taken together, evidence is provided that platelets provided by a single donor are sufficient to obtain PL for hASC propagation for clinical-scale applications mitigating the potential untoward side effects associated with the use of animal-derived reagents.

Correlation between mastitis occurrence and the count of microorganisms in bulk raw milk of bovine dairy herds in four selective culture media

Milk is the normal secretion of the mammary gland, practically free of colostrum and obtained by the complete milking of oil(? or more healthy animals. Mastitis is all inflammatory process of the mammary gland and it may cause alterations in the milk. The present work aimed to verify whether it is possible, by means of the counts of microorganism in the bulk raw milk ill four selective culture media, to establish a correlation with the occurrence of mastitis and therefore, to monitor this disease in bovine dairy herds. The following selective culture media were Used: KF Streptococcus Agar, Edwards Agar, Baird-Parker Agar, Blood Agar plus potassium tellurite. Spearman`s correlation coefficient was calculated in order to compare the Occurrence of mastitis (percentage) in each herd with respective selective culture media counts of microorganisms in bulk raw milk. Thirty-six possibilities were analysed (Tamis and CMT-positive rates were compared with the log-transformed count in four selective Culture media) and there was a negative correlation between Tamis 3 and the Baird-Parker Agar plate count. The total results of microbiological tests showed that there were three correlations of the counts in selective culture media. Fifty-two possibilities were analysed and there was a negative correlation between no-bacteria I-growth mastitis rates and log(10) of KF Streptoccocus Agar plate Count and there were two positive correlations between coagulase-positive staphylococci and log(10) of Baird-Parker Agar plate count and Blood Agar plus potassium tellurite plate count.

A critical comparison of the external and internal boron requirements for contrasting species in boron-buffered solution culture

Despite reports that boron (B) requirements differ among plant species there is a shortage of critical evidence to demonstrate unequivocally whether species differ in internal or external B requirements or both. The present research was conducted to establish the external and internal B requirements of three contrasting species, a woody dicot (marri), an herbaceous dicot (sunflower) and a monocot (wheat) using B-buffered solution culture. Boron-buffered solution culture provided satisfactory control of external B concentrations ranging from 0.04 to 30 muM throughout the 20- (sunflower and wheat) or 40-day (marri) growth period. At low external B concentrations (less than or equal to 0.13 muM), the growth of marri and sunflower was severely depressed but by contrast the vegetative growth of wheat plants was satisfactory and free of B deficiency symptoms. Marri and sunflower plants achieved total maximum shoot growth at greater than or equal to1.2 muM B in solutions while wheat plants did so at greater than or equal to 0.6 muM B. The critical B concentrations (mg kg(-1) dry matter) in the youngest open leaf blades of marri, sunflower and wheat plants were 17.9, 19.7 and 1.2 on 20, 10 and 10 days after transplanting (DAT), respectively. Lower internal and external B requirements of wheat were matched by a lower uptake rate of B compared to marri and sunflower.

Production of recombinant protein hLIF in static and dynamic systems of animal cell culture

Thesis for the Degree of Master of Science in Biotechnology Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

POTENTIALLY PATHOGENIC FREE-LIVING AMOEBAE IN SOME FLOOD-AFFECTED AREAS DURING 2011 CHIANG MAI FLOOD

SUMMARYThe survey was carried out to investigate the presence of potentially pathogenic free-living amoebae (FLA) during flood in Chiang Mai, Thailand in 2011. From different crisis flood areas, seven water samples were collected and tested for the presence of amoebae using culture and molecular methods. By monoxenic culture, FLA were detected from all samples at 37 °C incubation. The FLA growing at 37 °C were morphologically identified as Acanthamoeba spp., Naegleria spp. and some unidentified amoebae. Only three samples (42.8%), defined as thermotolerant FLA, continued to grow at 42 °C. By molecular methods, two non-thermotolerant FlA were shown to have 99% identity to Acanthamoeba sp. and 98% identity to Hartmannella vermiformis while the two thermotolerant FLA were identified as Echinamoeba exundans (100% identity) and Hartmannella sp. (99% identity). This first report of the occurrence of FLA in water during the flood disaster will provide information to the public to be aware of potentially pathogenic FLA.

Serological evidence for Saint Louis encephalitis virus in free-ranging New World monkeys and horses within the upper Paraná River basin region, Southern Brazil

Introduction Saint Louis encephalitis virus (SLEV) primarily occurs in the Americas and produces disease predominantly in humans. This study investigated the serological presence of SLEV in nonhuman primates and horses from southern Brazil. Methods From June 2004 to December 2005, sera from 133 monkeys (Alouatta caraya, n=43; Sapajus nigritus, n=64; Sapajus cay, n=26) trap-captured at the Paraná River basin region and 23 blood samples from farm horses were obtained and used for the serological detection of a panel of 19 arboviruses. All samples were analyzed in a hemagglutination inhibition (HI) assay; positive monkey samples were confirmed in a mouse neutralization test (MNT). Additionally, all blood samples were inoculated into C6/36 cell culture for viral isolation. Results Positive seroreactivity was only observed for SLEV. A prevalence of SLEV antibodies in sera was detected in Alouatta caraya (11.6%; 5/43), Sapajus nigritus (12.5%; 8/64), and S. cay (30.8%; 8/26) monkeys with the HI assay. Of the monkeys, 2.3% (1/42) of A. caraya, 6.3% 94/64) of S. nigritus, and 15.4% (4/26) of S. cay were positive for SLEV in the MNT. Additionally, SLEV antibodies were detected by HI in 39.1% (9/23) of the horses evaluated in this study. Arboviruses were not isolated from any blood sample. Conclusions These results confirmed the presence of SLEV in nonhuman primates and horses from southern Brazil. These findings most likely represent the first detection of this virus in nonhuman primates beyond the Amazon region. The detection of SLEV in animals within a geographical region distant from the Amazon basin suggests that there may be widespread and undiagnosed dissemination of this disease in Brazil.

Mechanical fatigue performance of PCL-chondroprogenitor constructs after cell culture under bioreactor mechanical stimulus

In tissue engineering of cartilage, polymeric scaffolds are implanted in the damaged tissue and subjected to repeated compression loading cycles. The possibility of failure due to mechanical fatigue has not been properly addressed in these scaffolds. Nevertheless, the macroporous scaffold is susceptible to failure after repeated loading-unloading cycles. This is related to inherent discontinuities in the material due to the micropore structure of the macro-pore walls that act as stress concentration points. In this work, chondrogenic precursor cells have been seeded in Poly-ε-caprolactone (PCL) scaffolds with fibrin and some were submitted to free swelling culture and others to cyclic loading in a bioreactor. After cell culture, all the samples were analyzed for fatigue behavior under repeated loading-unloading cycles. Moreover, some components of the extracellular matrix (ECM) were identified. No differences were observed between samples undergoing free swelling or bioreactor loading conditions, neither respect to matrix components nor to mechanical performance to fatigue. The ECM did not achieve the desired preponderance of collagen type II over collagen type I which is considered the main characteristic of hyaline cartilage ECM. However, prediction in PCL with ECM constructs was possible up to 600 cycles, an enhanced performance when compared to previous works. PCL after cell culture presents an improved fatigue resistance, despite the fact that the measured elastic modulus at the first cycle was similar to PCL with poly(vinyl alcohol) samples. This finding suggests that fatigue analysis in tissue engineering constructs can provide additional information missed with traditional mechanical measurements.

Seasonal variations in the intermediate metabolism of the crayfish Parastacus brasiliensis (Crustacea, Decapoda, Parastacidae) in the natural environment and experimental culture

The goal of this study was to evaluate the effects of seasonal variations on energy metabolism in different tissues of the freshwater crayfish Parastacus brasiliensis (von Martens, 1869). Crayfish were collected monthly from January 2001 to January 2003 in São Francisco de Paula, Rio Grande do Sul, Brazil, in a stream and in a culture tank. Haemolymph samples were collected from each crayfish in the field with a syringe, by puncturing the membrane at the base of the chelipeds. Hepatopancreas, gills, and abdominal muscle were removed for determination of free glucose, glycogen, total lipids, and triglycerides. The haemolymph samples were used for determination of glucose, total proteins, total lipids, and triglycerides. Statistical analysis revealed significant differences in biochemical composition in crayfish collected in the stream compared to the experimental tank during the year, principally in glucose and triglycerides in haemolymph, glycogen and total lipids in all tissues study, and triglycerides only in abdominal muscle. The regular food intake partially modified these seasonal variations of the metabolic pattern. Environmental conditions (e.g., food availability and water temperature) and reproductive period appeared to be the main factors influencing the seasonal patterns of variation in energy metabolism.