Time Of Cell Harvest Research Articles

A Novel Approach for Determining the Critical Quality Attributes of Mesenchymal Stem Cells by Specifying Cell Population With Replication Potential

We introduce a novel approach to determine the critical quality attributes (CQAs) of mesenchymal stem cells (MSCs) expected to exert immunosuppressive effects. MSCs retained homeostatic replication potentials, such as sustainable growth and consistent cell morphology as a population, in early passages, but lost them in late passages. Characteristic surface markers of MSCs (ie, CD73, CD90, and CD105) were no longer expressed at 2 weeks after subcutaneous transplantation into NOG mice when MSCs from late passages were transplanted, but not when MSCs from early passages were transplanted, suggesting that the biological effects of the MSCs differed according to the timing of cell harvesting and highlighting the importance of specifying MSCs that retained homeostatic features to define the CQAs. The homeostatic features of MSCs related to the balance of the redox system, nutrient requirements, and mitochondrial function were also observed until a certain passage. Therefore, we could define the CQAs of MSCs related to manufacturing by selecting process parameters (PPs) underlying the homeostatic features of MSCs and measuring these PPs quantitatively to specify the cell population with homeostatic features by limiting the passage number. The validity of the PPs stipulated in our pilot study was verified using an SKG murine arthritis model, and critical PPs (CPPs) were then selected among the PPs. Thus, CQAs related to manufacturing in the developmental phase could be defined by the CPPs in this manner, and the concept of CQAs could be refined continuously toward commercial manufacturing.

Efficient production of cellobionic acid using whole-cell biocatalyst of genetically modified Pseudomonas taetrolens.

Pseudomonas taetrolens has previously been shown to convert cellobiose to cellobionic acid (CBA), which can potentially be used in cosmetics, food, and pharmaceutical industries. The cellobiose-oxidizing activity of the P. taetrolens strain, which expressed the homologous quinoprotein glucose dehydrogenase (GDH), was increased by approximately 50.8% compared to the original strain. Whole-cell biocatalyst (WCB) of the genetically modified P. taetrolens strain [pDSK-GDH] was prepared simply by fermentation and washing processes. Reaction conditions for the proper use of WCB, such as reaction temperature, cell density to be added, and cell harvest time for preparing WCB, were investigated. The highest CBA productivity (18.2g/L/h) was achieved when WCB prepared in the late-exponential phase of cell culture was used at 35°C with cell density of 10 at OD600nm. Under these conditions, 200g/L of cellobiose was all converted to CBA in 11h, and the WCB of P. taetrolens [pDSK-GDH] maintained the maximum catalytic activity during at least six cycles without a significant decline in the productivity. Our results suggest that the manufacture of WCB based on genetically engineered P. taetrolens and its optimized use could be further developed as an economically viable option for the large-scale production of CBA.

Whole-cell biocatalysis using genetically modified Pseudomonas taetrolens for efficient production of maltobionic acid from pure maltose and high-maltose corn syrup.

Maltobionic acid (MBA) can be applied to various fields such as food, cosmetics, and pharmaceutical industries. In this study, whole-cell biocatalysis for MBA production was performed using recombinant Pseudomonas taetrolens homologously expressing quinoprotein glucose dehydrogenase (GDH). Various reaction parameters such as temperature, cell density, and cell harvest time, were optimized for improving MBA production. Under the optimized reaction conditions using pure maltose as a substrate, the MBA production titer, yield, and productivity of whole-cell biocatalyst (WCB) were 200g/L, 95.6%, and 18.18g/L/h, respectively, which were the highest compared to those reported previously. Productivity, a key factor for industrial MBA production, obtained from whole-cell biocatalysis in this study, was enhanced by approximately 1.9-fold compared to that obtained in our previous work (9.52g/L/h) using the fermentation method. Additionally, the WCB could be reused up to six times without a significant reduction in MBA productivity, indicating that the WCB is very robust. Although MBA productivity (8.33g/L/h) obtained from high-maltose corn syrup (HMCS) as a substrate was 45.8% of that using pure maltose, HMCS can be a better substrate for commercial MBA production because its price is only 1.1% of that of pure maltose. The results of this study using a WCB to convert maltose into MBA may support the development of a potential industrial process for more economically effective MBA production in the future.

Efficient production of lactobionic acid using genetically engineered Pseudomonas taetrolens as a whole-cell biocatalyst

Lactobionic acid (LBA) has been widely used in the food, pharmaceutical, and cosmetic industries. Pseudomonas taetrolens is an efficient LBA-producing bacterium. To improve the LBA-production ability of P. taetrolens, we modified the strain by genetic engineering. We performed homologous expression of the quinoprotein glucose dehydrogenase gene in P. taetrolens and measured the intracellular lactose-oxidizing activity and LBA production titer. In flask cultures at 12 h of incubation, the intracellular lactose oxidizing activity (0.159 U/g dry weight cell) and LBA production titer (77.2 g/L) of the recombinant P. taetrolens were approximately 118 % and 69 % higher than those (0.073 U/g dry weight cell and 45.8 g/L, respectively) of wild-type P. taetrolens. Using this recombinant strain as a whole-cell biocatalyst (WCB), the effects of reaction parameters, such as reaction temperature, cell density, and cell harvest time, were investigated on LBA production. Under optimized reaction conditions, the LBA production titer, yield, and productivity of WCB were 200 g/L, 95.6 %, and 16.7 g/L/h, respectively. Compared with our previous study, LBA production titer, yield, and productivity, which are key factors for industrial LBA production, were significantly improved by fermentation of wild-type P. taetrolens. Moreover, the reaction for LBA production could be performed up to seven times without a significant reduction in productivity, implying that this WCB was rather robust. Our results suggest that the utilization of whole-cell biocatalysis using recombinant P. taetrolens provides a potential solution to achieve economically feasible production of LBA.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model.

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

20-hydroxyecdysone accumulation and regulation in Ajuga lobata D. Don suspension culture.

Suspension culture of Ajuga lobata D. Don cells provides a method of synthesis of the phytoecdysteroid 20-hydroxyecdysone (20E) which can regulate the molting process of larvae. We characterized the culture conditions to optimize 20E production. Growth of A. lobata D. Don cells fits the logistic equation curve with a growth cycle of 19 days. Medium conductivity was negatively correlated with dry cell weight and 20E accumulation, thus could be used to determine the optimal time for cell harvest. Continuous subculture reduced 20E synthesis, but supplementing medium with 20E precursors mevalonic (MVA), α-Pinene, and nitric oxide (NO) can significantly promote cell growth and influence 20E accumulation. Combination of α-Pinene, MVA, and SNP significantly elevated 20E accumulation, thus may synergistically enhance 20E synthesis in A. lobata D. Don. The optimal concentrations of α-Pinene, MVA, and NO donor SNP in suspension culture were 50 μL L(-1), 10 mg L(-1), and 80 μmol L(-1).

Electrical Pulse Stimulation Induced Changes On Lipid, Mitochondrial, Glut4 And Ampk Content In Human Myotubes

It has been previously shown that human myotubes retain certain in vivo characteristics of the donors. Furthermore, we have shown that Electrical pulse stimulation (EPS), an in vitro exercise mimetic, increases mitochondrial and lipid content in cultured human myotubes after 24 hr. of stimulation. PURPOSE: We aimed to examine the EPS induced adaptations to lipid, mitochondrial, Glucose transporter 4 (GLUT4) and total 5’ Adenosine Monophosphate Activated Protein Kinase (AMPK) content using human myotubes from lean donors. Additionally, we investigated the impact of cell harvest time (immediate vs. 24 hr. after the EPS cessation) on the above-mentioned parameters. METHODS: EPS was applied to myotubes for 60 min (bipolar pulses of 100 Hz, 30V, every 5 seconds), 24 hr. or 48 hr. (single bipolar pulses of 1 Hz for 2 ms; 30V) and were harvested at two different time points: immediately after (early harvest) and 24hr after (late harvest) the end of stimulation. Mitochondrial and lipid content were measured by immunohistochemistry using primary antibody for complex IV and bodipy green dye, respectively. A microplate fluorometer was used to quantify fluorescence signals. GLUT4 and AMPK were measured using western immunoblotting. RESULTS: Lipid in myotubes was significantly higher (p<0.05) after 24 hr early harvest EPS compared to unstimulated control. There was no significant changes in lipid content with late harvest EPS. Mitochondrial content was significantly higher (p<0.05) after acute, 24 hr. as well as 48 hr. early harvest EPS, but was significantly lower (p<0.05) after acute late harvest EPS, compared to unstimulated controls. GLUT4 protein content was higher after acute and 24 hr. early harvest and 48 hr. late harvest EPS. Total AMPK was higher after 24 hr. early harvest and 48 hr. late harvest. AMPK was lower after 24 hr. late harvest. CONCLUSION: These findings suggest 24 hours of EPS treatment lead to increase in lipid, mitochondria, GLUT4 and AMPK protein content in human myotubes cultured from lean donors. Additionally, cell-harvesting time a reflection of post-exercise recovery is a key factor for these adaptations.

Are endothelial outgrowth cells a potential source for future re-vascularization therapy?

Endothelial progenitor cells (EPCs) represent a heterogeneous cell population that is believed to be involved in vasculogenesis after ischemic diseases. EPCs could have a potential for future therapies with the purpose of enhancing endothelial repair. However, due to the low amount of these cells in circulation they have to be expanded in vitro before administration into recipients. The purpose of this study was to analyse and evaluate possible changes in morphology and functionality as a result of in vitro ageing of a subtype of EPCs called endothelial outgrowth cells (EOCs), since such changes might compromise the cells' ability to participate in vasculogenesis.EOCs were isolated and grown from human umbilical cord blood using two methodologies with varying degree of cell purification. The changes between the two culture setups and the changes occurring in EOCs over time were traced by flow cytometry and assays for growth, tube formation, and beta-galactosidase production.The cells showed to be indistinguishable from each other during the first weeks of culture. The cells showed a changed morphology with bigger and more granular cells and the growth rate decreased with time. The cells also showed an increased Beta-galactosidase expression and decreased tube formation ability in late passage EOCs.Our data indicates that EOCs undergo senescence during long-term expansion and therefore time for cell harvest has to be validated in order to achieve functional cells still maintaining a therapeutic potential. A possible application in large animal or humans could be local injection of EOCs into affected areas and thereby reducing the need for long-term expansion of the cells.

Production of antibody fragment (Fab) throughout Escherichia coli fed-batch fermentation process: Changes in titre, location and form of product

Background: Recombinant proteins, including antibodies and antibody fragments, often contain disulfide bond bridges that are necessary for their folding, stability and function. Production of disulfide-bond-containing proteins in the periplasm of Escherichia coli has been very useful, due to unique characteristics of the periplasm, for obtaining fully active and correctly folded products and for alleviating downstream processing. Results: In this study, fed-batch cultivation of Escherichia coli (E. coli) for production of Fab D1.3, which is an anti-hen egg white Lysozyme (HEWL) antibody fragment was carried out at 37oC, and the bacterial cells were induced by adding 0.1 mM IPTG to the culture medium. Fermentor was sampled over the course of fermentation; the bacterial cells were centrifugally separated from the culture broth and subjected to osmotic shock (with excluding HEWL) and sonication procedures. The resulting fractions were analysed for Fab using a combination of ELISA, SDS-PAGE and Western blotting and changes in product titre, location, and form was assessed throughout growth. It was shown that osmotic shock released the Fab from the periplasm very efficiently and its efficacy was 20-45% more than sonication. This study demonstrates that, at high cell density cultivation in fermentor, target product can appear inside and outside the cells, depending on the time of induction. The maximum amount of Fab (47 mg/l) in the periplasm was reached at 14 hrs cultivation (4 hrs post induction), being suitable time for cell harvest, selective periplasmic extraction and downstream capture. The Fab increasingly leaked into the culture medium, and reached its maximum culture medium titre of ~78 mg/L after 6 hrs post induction. After 16 hrs cultivation (6 hrs post induction) the amount of Fab remained constant in different locations within and outside the cells. Western blot analysis of cell fractions showed that certain amount of the Fab was also produced in the cells as insoluble form. Conclusions: In this work we showed that the production of Fab in the periplasm during high cell density cultivation of E. coli in fermentor can be challenging as the product may appear in various locations within and outside the cells. To exploit the advantages of the periplasmic expression systems for purification in downstream processing, bacterial cells should be harvested when they maintain the majority of the target protein in their periplasmic space ( i.e. 4 hrs post induction).

Hyperglycemia induced and intrinsic alterations in type 2 diabetes-derived osteoclast function.

Periodontal disease-associated alveolar bone loss is a comorbidity of type-2-diabetes, where the roles of osteoclasts are poorly understood. To evaluate osteoclast differentiation and function in the context of type-2-diabetes. Bone marrow-derived osteoclasts from db/db mice, a model of type-2-diabetes, as well as human osteoclasts derived from peripheral blood of individuals with type-2-diabetes were evaluated for differentiation, resorption, and soluble mediator expression. While db/db mice were hyperglycemic at time of cell harvest, human participants were glycemically controlled. Although db/db cultures resulted in a higher number of larger osteoclasts, individual cell receptor activator of nuclear factor kappaB ligand (RANKL)-mediated bone resorption was similar to that observed in diabetes-free osteoclasts. Osteoclasts derived from individuals with type-2-diabetes differentiated similarly to controls with again no difference in bone resorbing capacity. Murine and human type-2-diabetes cultures both displayed inhibition of lipopolysaccharide (LPS)-induced deactivation and increased pro-osteoclastogenic mediator expression. Hyperglycemia plays a role in aberrant osteoclast differentiation leading to an increased capacity for bone resorption. Osteoclasts derived from murine models of and individuals with type-2-diabetes are unable to be inhibited by LPS, again leading to increased capacity for bone resorption. Here, environmental and intrinsic mechanisms associated with the increased alveolar bone loss observed in periodontal patients with type-2-diabetes are described.

Influence of Conditions on Reuterin Accumulation by the Resting Cell Biotransformation Process

3-Hydroxypropionaldehyde (3-HPA) is a potential valuable chemical and new broad-spectrum antimicrobial substance. In order to improve the conversion of 3-HPA/glycerol, our work studied the two-step process from glycerol to 3-HPA, and investigated the influence of cell harvest time, glycerol concentration, biomass concentration, pH and temperature on the production of 3-HPA by Lactobacillus reuteri CG001, respectively. The results showed that molar conversion yield of 3-HPA/glycerol reached 97.9% under the condition that 200 mmol·L −1 glycerol was converted by 25.3 g·L −1 resting cell for 1 h at 30°C. The cells could not be reused directly because the L. reuteri almost lost its bioconversion activity completely, but the ability of glycerol conversion could gradually recover if the fresh medium was added to the deactivated cell for 4 h.

The composition of the carbon source and the time of cell harvest are critical determinants of the final yield of human papillomavirus type 16 L1 protein produced in Saccharomyces cerevisiae

The L1 protein is a major component of prophylactic human papillomavirus (HPV) vaccines. Little effort has been made to improve the productivity of L1 protein by optimizing cell culture conditions although the high price of the vaccines is considered one of the factors limiting its widespread use. In biopharmaceutical manufacturing, strategies for optimizing culture conditions tend to focus on improvements in upstream processing rather than final yield because of the complexities of purification procedures. In this study, we investigated L1 protein productivity as a function of the composition of the carbon source and the point of cell harvesting in the Saccharomyces cerevisiae expression system. We performed 44 series of purifications and achieved the highest productivity when the cells were cultured in a medium composed of 7% glucose and 1% galactose for 144 h. The final yield of L1 protein was markedly affected by the glucose: galactose ratio and the point at which cells were harvested: there was a 15-fold difference between the lowest and highest yields. We believe that optimization of the composition of the carbon source and the time of cell harvest have considerable potential for reducing the cost of production of HPV L1 protein.

Glutathione Transport Is a Unique Function of the ATP-binding Cassette Protein ABCG2

Glutathione (GSH) transport is vital for maintenance of intracellular and extracellular redox balance. Only a few human proteins have been identified as transporters of GSH, glutathione disulfide (GSSG) and/or GSH conjugates (GS-X). Human epithelial MDA1586, A549, H1975, H460, HN4, and H157 cell lines were exposed to 2',5'-dihydroxychalcone, which induces a GSH efflux response. A real-time gene superarray for 84 proteins found in families that have a known role in GSH, GSSG, and/or GS-X transport was employed to help identify potential GSH transporters. ABCG2 was identified as the only gene in the array that closely corresponded with the magnitude of 2',5'-dihydroxychalcone (2',5'-DHC)-induced GSH efflux. The role of human ABCG2 as a novel GSH transporter was verified in a Saccharomyces cerevisiae galactose-inducible gene expression system. Yeast expressing human ABCG2 had 2.5-fold more extracellular GSH compared with those not expressing ABCG2. GSH efflux in ABCG2-expressing yeast was abolished by the ABCG2 substrate methotrexate (10 microM), indicating competitive inhibition. In contrast, 2',5'-DHC treatment of ABCG2-expressing yeast increased extracellular GSH levels in a dose-dependent manner with a maximum 3.5-fold increase in GSH after 24 h. In addition, suppression of ABCG2 with short hairpin RNA or ABCG2 overexpression in human epithelial cells decreased or increased extracellular GSH levels, respectively. Our data indicate that ABCG2 is a novel GSH transporter.

Characterization of the Metabolic Activation of Hepatitis C Virus Nucleoside Inhibitor β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) and Identification of a Novel Active 5′-Triphosphate Species

beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) replication in the subgenomic HCV replicon system, and its corresponding 5'-triphosphate is a potent inhibitor of the HCV RNA polymerase in vitro. In this study the formation of PSI-6130-triphosphate was characterized in primary human hepatocytes. PSI-6130 and its 5'-phosphorylated derivatives were identified, and the intracellular concentrations were determined. In addition, the deaminated derivative of PSI-6130, beta-d-2'-deoxy-2'-fluoro-2'-C-methyluridine (RO2433, PSI-6026) and its corresponding phosphorylated metabolites were identified in human hepatocytes after incubation with PSI-6130. The formation of the 5'-triphosphate (TP) of PSI-6130 (PSI-6130-TP) and RO2433 (RO2433-TP) increased with time and reached steady state levels at 48 h. The formation of both PSI-6130-TP and RO2433-TP demonstrated a linear relationship with the extracellular concentrations of PSI-6130 up to 100 mum, suggesting a high capacity of human hepatocytes to generate the two triphosphates. The mean half-lives of PSI-6130-TP and RO2433-TP were 4.7 and 38 h, respectively. RO2433-TP also inhibited RNA synthesis by the native HCV replicase isolated from HCV replicon cells and the recombinant HCV polymerase NS5B with potencies comparable with those of PSI-6130-TP. Incorporation of RO2433-5'-monophosphate (MP) into nascent RNA by NS5B led to chain termination similar to that of PSI-6130-MP. These results demonstrate that PSI-6130 is metabolized to two pharmacologically active species in primary human hepatocytes.

Regulatory cells develop after the spontaneous acceptance of rat liver allografts

After donor-specific transfusion, tolerance to heart transplants is serially passed to naive rats by the adoptive transfer of long-term survivor (LTS)-tolerant splenocytes (SC). We examined whether regulatory cells similarly develop after the spontaneously accepted Lewis (LEW) to Dark Agouti (DA) liver transplants. SC from a LTS DA rat with a LEW liver were adoptively transferred to a naive DA 1 day before transplantation of an irradiated (1000 rad) LEW liver. Untreated LEW to DA liver allografts were uniformly accepted; whereas all irradiated LEW liver grafts were rejected. In contrast, when 1.5 x 10 8 DA LTS SC were transferred to a naive DA recipient, all irradiated LEW liver grafts were accepted. When decreased to 1.0 x 10 8 LTS DA SC, only 1 of 4 irradiated LEW grafts was accepted. However, if 1.5 x 10 8 DA SC harvested only 30 days after liver transplantation were transferred, only 2 of 5 irradiated LEW liver grafts were accepted. The serial second and third adoptive transfers of 1.5 x 10 8 DA LTS SC also resulted in the uniform acceptance of irradiated LEW livers. Regulatory cells that develop after the spontaneous acceptance of a LEW to DA liver transplant can serially transfer tolerance to new naive LEW liver allograft DA recipients. This "infectious tolerance" is dependent on the time of cell harvest after transplantation and on the cell dose given.

123. Adenovirus Type 11 Based Vectors as a Platform for Treatment of Metastatic and Hematopoietic Disease

Adenovirus (Ad) vectors derived from group C serotype Ad5 have shown great promise for treatment of disease based on their ability to efficiently infect multiple therapeutic target cell populations. Despite this ability these vector systems do not efficiently infect all potential disease target cell types, due to a lack of primary attachment receptors on target cells, and are hampered in vivo by native virus neutralizing immune responses and acute vector-mediated toxicity. In an attempt to circumvent these limitations to Ad-mediated gene therapy we have developed a vector system based on group B serotype Ad11 whereby first generation (E1 deleted) vector genomes can be generated by E.coli recombination or by co-transfection of 2 shuttle plasmids into the cell line 293-Ad11E1B55K. Vector preparations of > 1012 particles/ml can be generated with the optimum cell harvest time being 72 hours after infection. Competition experiments using recombinant Ad11p fiber knob to block binding of the vector Ad11-CMV-GFP to CHO cells expressing the previously identified adenovirus receptors CAR and CD46 demonstrated that Ad11 vectors are able to infect cells through a CAR-independent fiber knob/CD46 mediated pathway. Analysis of serum samples taken from normal US or normal and cervical cancer positive Senegalese volunteers demonstrated that virus directed neutralizing antibodies (VNAbs) against Ad11 were present in less than 10% of samples whilst VNAbs against Ad5 were present in more than 30% of samples. Transformation assays in baby rat kidney (BRK) cells transfected with wild type or E1 deleted genomes from Ad5 or Ad11 showed no transformed foci in E1 deleted genome transfected cells of either serotype whilst wild type Ad5 showed more foci than wild type Ad11. Transduction studies using Ad5 and Ad11 vectors expressing the reporter gene GFP from the CMV promoter were also carried out. A panel of human tumor cell lines derived from cervix, lung, liver, breast, prostate, colon, ovary, blood and brain demonstrated transduction profiles for Ad5 and Ad11 derived vectors that correlated with levels of CAR and CD46 expression. Primary human cord blood derived CD34+ve cells and peripheral blood derived dendritic cells were readily infectable with Ad11 derived vectors in contrast to vectors based on Ad5. Additionally we previously developed an Ad5 based system (Ad.IR) in which tumor specific replication of E1 deleted Ad DNA upon infection mediates expression of a toxic protein. Replication of Ad11 vector DNA in a panel of tumor cells demonstrated the suitability of Ad11 based vectors for application in an Ad.IR system. In summary we have developed E1 deleted Ad11 vectors that can utilize CD46 for cell infection, have a low prevalence to neutralization by human serum and are non-transforming. These vectors provide an excellent platform for the treatment of diseases in which Ad5 based vectors have shown limitations including treatment of metastatic and hematopoietic disease.

Circadian rhythm of cytokine secretion following thermal injury in mice: implications for burn and trauma research.

Although there are many reports of circadian variation in hormone secretion, there are only a few reports on the relationship between circadian rhythm and cytokine production. The aim of the present studies was to investigate whether there is a circadian effect on cytokine production of splenic lymphocytes and adherent splenocytes in mice after burn or sham injury. We selected day 7 after injury for our determinations because we have previously shown day 7 is the time of maximal suppression of T cell IL-2 and IFNgamma production and maximal increase in adherent cell proinflammatory cytokine secretion in this model. IL-2 and TNFalpha were chosen as reference cytokines since the former is known to be produced by T cells and the latter by adherent cells of the innate immune system. The results showed that seven days after sham or thermal injury both T cell IL-2 and adherent cell TNFalpha production were altered by time of injury or time of cell harvest. IL-2 secretion was significantly decreased in burn compared to sham animals when splenocytes were harvested in the morning; the decrease was non-significant when splenocytes were harvested in the afternoon. TNFalpha secretion was significantly increased in burn vs. sham adherent cells only when injury took place in the morning. The observed circadian variations in cytokine production could have a significant effect on cytokine levels measured in clinical and animal studies of injury and may explain some of the reported discrepancies among these studies.

Human Pluripotent and Progenitor Cells Display Cell Surface Cluster Differentiation Markers CD10, CD13, CD56, and MHC Class-I

Each year millions of people suffer tissue loss or end-stage organ failure. While allogeneic therapies have saved and improved countless lives, they remain imperfect solutions. These therapies are limited by critical donor shortages, long-term morbidity, and mortality. A wide variety of transplants, congenital malformations, elective surgeries, and genetic disorders have the potential for treatment with autologous stem cells as a source of HLA-matched donor tissue. Our current research is aimed at characterizing cell surface cluster differentiation (CD) markers on human progenitor and pluripotent cells to aid in isolating comparatively purified populations of these cells. This study examined human pluripotent and progenitor cells isolated from fetal, mature, and geriatric individuals for the possible presence of 15 CD markers. The response to insulin and dexamethasone revealed that the cell isolates were composed of lineage-committed progenitor cells and lineage-uncommitted pluripotent cells. Flow cytometry showed cell populations positive for CD10, CD13, CD56, and MHC Class-I markers and negative for CD3, CD5, CD7, CD11b, CD14, CD15, CD16, CD19, CD25, CD45, and CD65 markers. Northern analysis revealed that CD13 and CD56 were actively transcribed at time of cell harvest. We report the first identification of CD10, CD13, CD56, and MHC Class-I cell surface antigens on these human cells.

Human pluripotent and progenitor cells display cell surface cluster differentiation markers CD10, CD13, CD56, and MHC class-I.

Each year millions of people suffer tissue loss or end-stage organ failure. While allogeneic therapies have saved and improved countless lives, they remain imperfect solutions. These therapies are limited by critical donor shortages, long-term morbidity, and mortality. A wide variety of transplants, congenital malformations, elective surgeries, and genetic disorders have the potential for treatment with autologous stem cells as a source of HLA-matched donor tissue. Our current research is aimed at characterizing cell surface cluster differentiation (CD) markers on human progenitor and pluripotent cells to aid in isolating comparatively purified populations of these cells. This study examined human pluripotent and progenitor cells isolated from fetal, mature, and geriatric individuals for the possible presence of 15 CD markers. The response to insulin and dexamethasone revealed that the cell isolates were composed of lineage-committed progenitor cells and lineage-uncommitted pluripotent cells. Flow cytometry showed cell populations positive for CD10, CD13, CD56, and MHC Class-I markers and negative for CD3, CD5, CD7, CD11b, CD14, CD15, CD16, CD19, CD25, CD45, and CD65 markers. Northern analysis revealed that CD13 and CD56 were actively transcribed at time of cell harvest. We report the first identification of CD10, CD13, CD56, and MHC Class-I cell surface antigens on these human cells.

Large-scale production of Mandelate racemase by Pseudomonas putida ATCC 12633: optimization of enzyme induction and development of a stable crude enzyme preparation

The production of mandelate racemase [EC 5.1.2.2] from Pseudomonas putida ATCC 12633 was optimized with respect to (i) biomass and (ii) enzyme induction. The dependence of biomass yield and enzyme content on the concentration of carbon-sources (such as glucose and mandelate) was determined by applying batch- and fed-batch cultivation on a ten-liter scale. On the one hand, highest cell-densities were obtained in fed-batch cultivations with repeated glucose- and mandelate-feed but mandelate racemase induction was low. On the other hand, excellent enzyme-induction was obtained in batch-cultivation when both glucose and mandelate were used right from the start as carbon source. The optimal time for cell-harvest was indicated by a characteristic decolorization of the growth medium from grey/blue to yellow. This latter phenomenon (which is caused by the degradation of the blue Fe(III)-complex of catechol, a late intermediate of the mandelate pathway) greatly simplified the monitoring of the enzyme activity and the determination of the optimal time for harvest. Based on these results, a lyophilized crude enzyme preparation was prepared from a 10-liter bioreactor showing a total specific activity of 23 10 6 U ( μmol g −1 min −1). This represents roughly a 300-fold enhancement in activity as compared to the previous protocol.