Platelets from mice lacking the aryl hydrocarbon receptor exhibit defective collagen-dependent signaling.

BACKGROUND: We previously identified aryl hydrocarbon receptor (AHR) as a novel regulator of megakaryocytic differentiation and polyploidization and reported that AHR-null mice have approximately 15% fewer platelets than do wild-type mice, yet they exhibit a dramatic, unexplained bleeding phenotype. OBJECTIVES: The current work tests our hypothesis that AHR-null platelets are functionally deficient, contributing to the previously reported (yet unexplained) bleeding phenotype present in AHR-null mice. METHODS: AHR-null bone marrow was ex vivo differentiated with thrombopoietin with or without AHR ligands or AHR inhibitors and analyzed for degree of megakaryopoiesis and polyploidization. Platelet function of AHR-null mice was assessed with aggregation and spreading assays. Platelet signaling was examined using Western analysis and Rac activity assays. RESULTS: AHR ligands differentiate murine bone marrow-derived progenitors into polyploid megakaryocytes in the absence of thrombopoietin, and AHR inhibitors block thrombopoietin-induced megakaryocytic differentiation. Despite their responsiveness toward thrombin, AHR-null platelets demonstrate decreased aggregation and spreading in response to collagen compared with wild-type platelets. AHR-null platelets bind fibrinogen after stimulation with thrombin or AYPGKF and aggregate in response to AYPGKF and adenosine diphosphate. Mechanistically, AHR absence led to down-regulation of Vav1 and Vav3, altered phospholipase Cγ2 phosphorylation, decreased Rac1 activation, and reduced platelet activation in response to collagen. CONCLUSIONS: These results are consistent with a role for AHR in platelet function, especially as it relates to platelet aggregation and spreading in response to collagen. Our work suggests AHR is a critical component of the physiologic response that platelets undergo in response to collagen and may provide novel treatment options for patients with bleeding disorders.

Higher pH promotes megakaryocytic maturation and apoptosis.

Megakaryocytic (Mk) cells mature adjacent to bone marrow (BM) sinus walls and subsequently release platelets within the sinusoidal space or in lung capillaries. In contrast, primitive stem and Mk progenitor cells reside the furthest away from the BM sinus walls. The existence of pH gradients in the BM raises the question of whether pH affects Mk maturation and differentiation. We generated Mk cells from peripheral blood CD34(+) cells in a serum-free medium at different pH levels (7.2, 7.4, and 7.6) and found that higher pH resulted in an earlier and higher polyploidization of CD41(+) Mk cells and an earlier onset of Mk-cell apoptosis. The peak day of high ploidy was correlated well with the onset day of Mk apoptosis, thus suggesting that a decline in the fraction of high-ploidy Mk cells at the late culture stage is caused by Mk-cell apoptosis. We further explored the relationship between Mk-cell maturation and apoptosis by employing an antiapoptotic agent Z-Val-Ala-Asp(Ome)-FMK (zVAD). Addition of zVAD led to an average 30% higher and 2.8-day delayed polyploidization, while apoptosis was delayed by 2.4 days. Faster depletion of CD34(+) cells and an earlier peak in the fraction of larger colony-forming Mk cells (BFU-Mks) were also observed at higher pH. Taken together, these data suggest that higher pH promotes Mk-cell differentiation, maturation, and apoptosis.

Oxygen tension modulates the expression of cytokine receptors, transcription factors, and lineage-specific markers in cultured human megakaryocytes.

OBJECTIVE: We have recently reported that 20% O2 significantly enhances total megakaryocyte (Mk) number, polyploidy, and proplatelet formation compared to 5% O2 in culture. In order to further elucidate the regulatory role of pO2 on megakaryocytopoiesis, we conducted a kinetic study of the expression of surface markers CD41a and CD42a; receptors for thrombopoietin (TPO), interleukin-3 (IL-3), and Flt3-ligand; the glutamate receptor of the N-methyl-D-aspartate subtype 1 (NMDAR1); and transcription factors GATA-1, NF-E2, and E2F-1. MATERIALS AND METHODS: Mks were generated from mobilized peripheral blood (PB) CD34+ cells from normal donors in serum-free medium with TPO, IL-3, and Flt3-ligand at 20% and 5% O2. Quantitative assessment of Mk surface receptors and nuclear transcription factors was performed using multiparameter flow cytometry. mRNA levels of the nuclear transcription factors GATA-1 and NF-E2 were evaluated using RT-PCR. RESULTS: The proportions of cells expressing the early Mk marker CD41a and the late Mk marker CD42a at day 15 were 4 and 5 times higher, respectively, at 20% O2. CD41a and CD42a protein levels per cell were also higher at 20% O2. After day 5, c-Mpl (TPO receptor) generally followed similar kinetics as CD41a. The proportion of IL-3 receptor (IL-3R)++ Mks at day 5 was 1.5 times higher at 5% O2. The NMDAR1 protein previously known to be expressed by neuronal cells has recently been identified in Mks. NMDAR1 and the transcription factors were studied on days 6, 9, and 11. NMDAR1 was expressed at a 1.5- to 1.8-fold higher level at 5% O2. Twenty percent O2 supported higher expression of the Mk-early and -late-maturation-specific transcription factors GATA-1 (1.2- to 2.2-fold higher) and NF-E2 (1.1- to 2.8-fold higher). This was consistent with RT-PCR data indicating the presence of higher levels of GATA-1 and NF-E2 mRNA at 20% O2. E2F-1, a ubiquitously expressed cell cycle transcription factor, was expressed at a 1.5-fold higher level at 20% O2 on day 6, but this difference did not persist by day 9. CONCLUSION: These findings demonstrate that cytokine receptors c-Mpl and IL-3R, and Mk differentiation-specific surface receptors CD41a, CD42a, and NMDAR1, are significantly modulated by pO2, and suggest that one of the mechanisms of enhanced maturation at 20% O2 may involve regulation of transcription factors GATA-1 and NF-E2.

Modeling pO(2) distributions in the bone marrow hematopoietic compartment. I. Krogh's model.

Human bone marrow (BM) is a tissue of complex architectural organization, which includes granulopoietic loci, erythroblastic islets, and lymphocytic nodules. Oxygen tension (pO(2)) is an important determinant of hematopoietic stem and progenitor cell proliferation and differentiation. Thus, understanding the impact of the BM architectural organization on pO(2) levels in extravascular hematopoietic tissue is an important biophysical problem. However, currently it is impossible to measure pO(2) levels and their spatial variations in the BM. Homogeneous Kroghian models were used to estimate pO(2) distribution in the BM hematopoietic compartment (BMHC) and to conservatively simulate pO(2)-limited cellular architectures. Based on biophysical data of hematopoietic cells and characteristics of BM physiology, we constructed a tissue cylinder solely occupied by granulocytic progenitors (the most metabolically active stage of the most abundant cell type) to provide a physiologically relevant limiting case. Although the number of possible cellular architectures is large, all simulated pO(2) profiles fall between two extreme cases: those of homogeneous tissues with adipocytes and granulocytic progenitors, respectively. This was illustrated by results obtained from a parametric criterion derived for pO(2) depletion in the extravascular tissue. Modeling results suggest that stem and progenitor cells experience a low pO(2) environment in the BMHC.

Modeling pO(2) distributions in the bone marrow hematopoietic compartment. II. Modified Kroghian models.

Hematopoietic cells of various lineages are organized in distinct cellular architectures in the bone marrow hematopoietic compartment (BMHC). The homogeneous Kroghian model, which deals only with a single cell type, may not be sufficient to accurately describe oxygen transfer in the BMHC. Thus, for cellular architectures of physiological significance, more complex biophysical-transport models were considered and compared against simulations using the homogeneous Kroghian model. The effects of the heterogeneity of model parameters on the oxygen tension (pO(2)) distribution were examined using the multilayer Kroghian model. We have also developed two-dimensional Kroghian models to simulate several cellular architectures in which a cell cluster (erythroid cluster) or an individual cell (megakaryocyte or adipocyte) is located in the BMHC predominantly occupied by mature granulocytes. pO(2) distributions in colony-type cellular arrangements (erythroblastic islets, granulopoietic loci, and lymphocytic nodules) in the BMHC were also evaluated by modifying the multilayer Kroghian model. The simulated results indicate that most hematopoietic progenitors experience low pO(2) values, which agrees with the finding that low pO(2) promotes the expansion of various hematopoietic progenitors. These results suggest that the most primitive stem cells, which are located even further away from BM sinuses, are likely located in a very low pO(2) environment.

Model-based estimation of myeloid hematopoietic progenitor cells in ex vivo cultures for cell and gene therapies.

Ex vivo production of hematopoietic progenitor cells has potential applications for cell therapy to alleviate cytopenias associated with chemotherapy and for gene therapy. In both therapies, progenitor and stem cells are considered crucial factors for therapeutic success. Assays for progenitor cells, however, take 2 weeks to complete, which is similar to the length of a typical culture. Therefore, a real-time estimation of the percentage or number of progenitor cells, based on rapid measurements, would be useful for optimization of feeding and harvest decisions. In this study, metabolic activity assays and flow cytometric analysis were used to estimate the content of progenitor cells. The measured metabolic activities are a collective contribution from all types of cells. Cells in granulomonocytic cultures have been lumped into six cell types and metabolic rates have been modeled as a linear function of cell composition and growth rate and as a nonlinear function of cell density. Data from 24 experiments were utilized to determine the model parameters in a calibration step. These data include flow cytometric analysis of more mature hematopoietic cells, progenitor cell colony assays, total cell content, and metabolite concentrations, and cover a wide range of cell composition, cell density, and growth rate. After calibration, the model is able to deliver good predictions of progenitor cell content for cultures with higher percentages of progenitor cells, as well as the peak progenitor cell content, based only on parameters that can be rapidly measured. With the aid of those predictions a harvest strategy was developed that will allow optimizing the harvest time based on the culture kinetics of each patient or donor inoculum, rather than using retrospective analysis to determine a uniform harvest time.

Low oxygen tension and autologous plasma enhance T-cell proliferation and CD49d expression density in serum-free media.

BACKGROUND: As cellular immunotherapy with ex vivo expanded cells becomes more widely used to treat a variety of illnesses, optimization of culture parameters, to maximize cell production and function, is essential for continued success. The effects of reduced oxygen tension and autologous plasma on T-cell expansion, receptor expression, apoptosis, and cytolytic activity in serum-free media were investigated. METHODS: PBMCs derived from whole blood samples were activated with anti-CD3 and anti-CD28 MAb in serum-free (AIM V) medium containing IL-2, and maintained at 5% and 20% oxygen tension. In some cases cultures were supplemented with 2% autologous plasma. RESULTS: Low oxygen enhanced T-cell expansion 13- and 4.8-fold in serum-free and plasma-supplemented media, respectively. Autologous plasma also had a beneficial effect on T-cell cultures. Plasma-supplemented cultures expanded 74-fold more than serum-free cultures at low oxygen tension, and 43-fold more at high oxygen tension. Several samples expanded very poorly under serum-free conditions, and reasonable cell numbers were obtained only from plasma-supplemented cultures. CD49d expression density increased 3-fold to 4-fold in cultures supplemented with plasma. In contrast to our previous findings in serum-containing media, IL-2 receptor expression kinetics were unaffected by oxygen tension. No effects caused by oxygen tension or autologous plasma on expression of other surface antigens (CD4, CD8, CD44, CD95) were observed. DISCUSSION: Low oxygen tension and autologous plasma greatly increase expansion of T cells, thereby decreasing the time needed for production of cells for prophylaxis. Increased CD49d expression density may translate into improved migration and cytotoxicity.

Fermentation characterization and flux analysis of recombinant strains of Clostridium acetobutylicum with an inactivated solR gene.

The effect of solR inactivation on the metabolism of Clostridium acetobutylicum was examined using fermentation characterization and metabolic flux analysis. The solR-inactivated strain (SolRH) of this study had a higher rate of glucose utilization and produced higher solvent concentrations (by 25%, 14%, and 81%, respectively, for butanol, acetone, and ethanol) compared to the wild type. Strain SolRH(pTAAD), carrying a plasmid-encoded copy of the bifunctional alcohol/aldehyde dehydrogenase gene (aad) used in butanol production, produced even higher concentrations of solvents (by 21%, 45%, and 62%, respectively, for butanol, acetone, and ethanol) than strain SolRH. Clarithromycin used for strain SolRH maintenance during SolRH(pTAAD) fermentations did not alter product formation; however, tetracycline used for pTAAD maintenance resulted in 90% lower solvent production.

Oxygen tension influences the differentiation, maturation and apoptosis of human megakaryocytes.

Megakaryocytes (Mks) mature adjacent to bone marrow (BM) sinus walls and subsequently release platelets within the sinusoidal space or in lung capillaries. As the sites for platelet release have higher levels of oxygen tension (pO(2)) than the core of the BM where stem and progenitor cells reside, we investigated whether pO(2) influences Mk maturation. Mks were generated from CD34(+) cells (from mobilized peripheral blood from cancer patients) under 5% and 20% O(2). At day 15, CD41(+) Mk expansion in 20% and 5% O(2) cultures was 85-fold and 31-fold respectively. Twenty percent O(2) cultures also had higher levels of high ploidy (> or = 8N, eightfold higher) and proplatelet-forming (fivefold higher) Mks. At day 21, 20% O(2) cultures had a fivefold higher number of apoptotic Mks. In contrast, 5% O(2) promoted Mk colony-forming unit (CFU-Mk) generation and maintenance. Similar results were observed in cultures initiated with CD41(+) Mks, indicating that pO(2) directly affects Mks. The change from 20% to 5% O(2) on day 5 and day 7 delayed both maturation and apoptosis, suggesting that these two processes are closely linked. These results were confirmed in CD34(+) cultures from normal BM samples. These data may provide insights into in vivo Mk maturation, such as an explanation for hypoxia-induced thrombocytopenia in animals.

The lactate issue revisited: novel feeding protocols to examine inhibition of cell proliferation and glucose metabolism in hematopoietic cell cultures.

It is well established that cell proliferation in batch (unfed) hematopoietic cell cultures is greatly inhibited relative to that in cultures with feeding. What is not known, however, is the nature of this inhibition. On the basis of our observations in hematopoietic cultures that cell proliferation ceases when the lactate concentration ([lactate]) exceeds 20 mM (accompanied by a decrease in culture pH), we investigated the effect of lactate accumulation on cell proliferation, metabolism, and differentiation. We differ in our approach from previous efforts in that we have tried to more accurately recreate the manner in which lactate accumulates in culture by employing a daily feeding protocol in which [lactate] and/or pH in the fresh medium was adjusted to match the conditions prior to feeding. We conclude that the decrease in pH associated with lactate accumulation significantly inhibits both cell proliferation and metabolism. Although inhibition in cultures with high [lactate] and low pH is similar to that in unfed cultures, pH control in unfed cultures does not alleviate the inhibition, indicating that other inhibitory factors are also present. Thus, pH control is necessary, but not sufficient, to eliminate inhibition of cell growth and metabolism in unfed hematopoietic cell cultures. We also conclude that high [lactate] and low pH have little effect on cell differentiation in fed cultures, although there is evidence to suggest that low pH may play a role in monocyte differentiation in unfed cultures.

Dynamic model of ex vivo granulocytic kinetics to examine the effects of oxygen tension, pH, and interleukin-3.

OBJECTIVE: Evaluating kinetics in hematopoietic cultures is complicated by the distribution of cells over various stages of differentiation and by the presence of cells from different lineages. Thus, an observed response is an integral response from distributed cell populations. Growth factors and other parameters can greatly affect the lineage and maturation stage of the culture outcome. To resolve the kinetics and more clearly define the differential effects of O(2) tension (pO(2)), pH, and interleukin-3 (IL-3) on granulopoiesis, a mathematical model-based approach was undertaken. MATERIALS AND METHODS: Granulocytic differentiation is described within a continuous, deterministic framework in which cells develop from primitive granulocytic progenitors to mature neutrophils. The model predicts two distributed populations-quiescent and cycling cells-by incorporating rates of growth, death, differentiation, and transition between quiescence and active cycling. The response of these four model processes to changes in the culture environment was examined. RESULTS: Model simulations of experimental data revealed the following: 1) pO(2) effects are exerted only on the growth rate but not maturation times. 2) pH effects between pH 7.25 and 7.4 on growth and differentiation are coupled; however, with increasing pH values, especially at pH 7. 6, the death rate for cells in the early stages of differentiation becomes increasingly significant. 3) The absence of IL-3 increases the death rate for primitive cells only minimally but markedly enhances the rate of differentiation through the myeloblast window in the differentiation pathway. The combined effects of these environmental factors can be predicted based on changes in the model parameters derived from the individual effects. CONCLUSIONS: Experimental data combined with mathematical modeling can elucidate the mechanisms underlying the regulation of granulopoiesis by pO(2), pH, and IL-3. The model also can be readily adapted to evaluate the effects of other culture conditions. The increased understanding of experimental results gained with this approach can be used to modify culture conditions to optimize ex vivo production of neutrophil precursors.

Culture of human T cells in stirred bioreactors for cellular immunotherapy applications: shear, proliferation, and the IL-2 receptor.

Ex vivo expansion of T cells is a key step of many cellular immunotherapy protocols, which require large numbers of immune cells to eradicate malignant or virally infected cells. The use of stirred culture systems for T cell expansion offers many potential advantages over the static culture systems commonly used today, including homogeneity of culture conditions, ease of sampling, and implementation of control systems. Primary human T cells as well as the transformed TALL103/2 T cell line were cultured in 100-mL spinner flasks as well as 2-L bioreactors to investigate the effects of shear forces produced by agitation and sparging-based aeration on the expansion of T cells. Primary T cells could be successfully grown at agitation rates of up to 120 rpm in the spinner flasks and to 180 rpm in the bioreactors with no immediate detrimental effects on proliferation. Exposure to agitation and sparging did, however, cause a significantly increased rate of downregulation of the interleukin-2 receptor (IL-2R), resulting in lower overall expansion potential from a single stimulation as compared to static controls, with faster IL-2R downregulation occurring at higher agitation rates. For the primary T cells, no significant effects of agitation were found on expression levels of other key surface receptors (CD3, CD28, or CD62L) examined. No significant effects of agitation were observed on primary T cell metabolism or levels of cellular apoptosis in the cultures. The TALL103/2 T cell line was found to be extremely sensitive to agitation, showing severely reduced growth at speeds above 30 rpm in 100-mL spinner flasks. This unexpected increased fragility in the transformed T cell line as compared to primary T cells points out the importance of carefully selecting a model cell line which will accurately represent the characteristics of the cell system of interest.

Equations and calculations for fermentations of butyric acid bacteria. Reprinted from Biotechnology and Bioengineering, Vol. XXVI, Pp 174-187 (1984).

A stoichiometric equation has been derived which describes the interrelations among the various products and biomass in fermentations of butyric acid bacteria. The derivation of the equation is based on an assumed ATP yield, two biological regularities, and the biochemistry of product formation of the fermentations. The equation obeys the constraints imposed on growth and product formation by thermodynamics and the biochemical topology. The validity of the equation is tested using a variety of fermentation data from the literature. The uses, improvements, limitations, and extensions of the equation are also discussed in detail. For example, the fermentation equation is used to calculate the maximal possible yields of the main fermentation products.

Physiologically significant effects of pH and oxygen tension on granulopoiesis.

OBJECTIVE: Granulocyte differentiation in the bone marrow (BM) takes place in regions with lower pH and O(2) tension (pO(2)) than those in the BM sinuses. This suggests that granulopoiesis will be enhanced at subvascular pH and pO(2). MATERIALS AND METHODS: The effects of pH AND pO2 on granulocyte proliferation, differentiation, and granulocyte colony-stimulating factor receptor (G-CSFR) expression were evaluated using mobilized peripheral blood CD34(+) cells directed down the granulocytic pathway with stem cell factor, interleukin 3, interleukin 6, and G-CSF. RESULTS: Cell expansion was enhanced at subvascular pH, with twice as many total cells and CD15(bright)/CD11b(+) late neutrophil precursors (myelocytes, metamyelocytes, bands) produced at pH 7.07 to 7.21 as was produced at pH 7.38. Low pH accelerated the rate of differentiation concomitant with this increase in proliferation. Also, total, CD15(bright)/CD11b(-) (promyelocytes, early myelocytes), and CD15(bright)/CD11b(+) cell expansion was enhanced at lower pO(2), with twice as many of each cell type produced at 5% O(2) as at 20% O(2). The effects of low pH and low pO(2) were additive, such that generation of total, CD15(bright)/CD11b(-), and CD15(bright)/CD11b(+) cells was 3.5-, 2.4-, and 4.0-fold greater at pH 7.21 and 5% O(2) than at the standard hematopoietic culture conditions of pH 7.38 and 20% O(2). Low pH resulted in faster upregulation of G-CSFR surface expression, whereas pO(2) had no effect on G-CSFR expression. CONCLUSION: These data provide compelling evidence that pH and pO(2) gradients within the BM play significant roles in regulating hematopoiesis. More rapid granulocytic cell proliferation and differentiation at low pH may be explained in part by more rapid G-CSFR expression. The ability to alter cell development by manipulating pH and pO(2) has important implications for optimizing ex vivo production of neutrophil precursors.

Extracellular pH affects the proliferation of cultured human T cells and their expression of the interleukin-2 receptor.

Ex vivo expansion of T cells is an important aspect of many cellular immunotherapy protocols, and the effects of the culture environment on the cells must be understood to produce large numbers of functional cells. Extracellular pH is a fundamental parameter that has many different effects on cultured cells. In this study, peripheral blood mononuclear cells were stimulated with phytohemagglutinin and cultured at pH values of 7.0, 7.2, or 7.4. The effects of pH on the cells were studied during the 2 to 3 weeks of proliferation resulting from phytohemagglutinin stimulation, in order to examine the culture kinetics over realistic time scales for ex vivo expansion. The proliferation capacity of the T cells increased more than three-fold for the pH 7.0 and 7.2 cultures compared with the pH 7.4 cultures. The culture pH also affected the kinetics of the interleukin-2 receptor down-regulation process. The faster receptor down-regulation in both the pH 7.2 and 7.4 cultures resulted in a more than twofold greater fraction of interleukin-2 receptor(+) cells in the pH 7.0 cultures. Although the fraction of apoptotic cells (using the Annexin V flow-cytometric method) remained less than 10%, we observed 27% more apoptosis in the pH 7.4 cultures than in the 7.2 cultures and 49% more apoptosis in the pH 7.4 cultures than in the 7.0 cultures. These effects on interleukin-2 receptor expression and cellular apoptosis may partially explain the observed effects of pH on T-cell proliferation.

Cell density-dependent proliferation in frequently-fed peripheral blood mononuclear cell cultures.

BACKGROUND: Our goal was to produce granulocyte progenitor (CFU-G) and post-progenitor (CD15(+)CD11b(+/-)) cells for subsequent transplantation. We hypothesized that increasing the feeding frequency and maintaining constant densities may overcome inhibitory growth conditions (i.e. low pH) in high-density cultures. METHODS: To study the effect of cell density on total cell expansion, differentiation and lactate production, 50% daily medium exchanges were used in cultures of peripheral blood mononuclear cells (PB MNC) maintained at constant densities (ranging from 5 x 10(4)cells/mL to 2.5 x 10(6)cells/mL). RESULTS: We observed a significant increase in total cell expansion when the density was increased from 5 x 10(4) cells/mL to 1 x 10(6) cells/mL, but a further increase to 2.5 x 10(6)cells/mL resulted in a decline in cell expansion. Increasing feeding to 90% daily exchange in cultures with 2.5 x 10(6) cells/mL did not enhance cell expansion; nor did reducing the extent of feeding in cultures with 5 x 10(4) cells/mL to 10% daily exchange. We did not observe a relationship between cell density and the percentage of granulocyte progenitor and post-progenitor (CD15(+)CD11b(-/+)) cells. While specific lactate production (q(lac)) in cultures with 2.5 x 10(6) cells/mL was approximately 60% of those observed in lower density cultures by Day 13, this difference was largely eliminated by increasing the extent of feeding in cultures with 2.5 x 10(6) cells/mL. DISCUSSION: Our results suggest that feeding rates must be adjusted according to cell density to maximize culture performance. They also suggest that cellular crowding on the culture surface can limit expansion in suspension (nonadherent) cultures.

Clinical-scale production of granulocyte progenitor and post-progenitor cells using daniplestim, leridistim, Progenipoietin, Promegapoietin and autologous plasma.

BACKGROUND: Supplementation of PBPC autografts with ex vivo expanded PBMC may significantly reduce or eliminate the period of neutropenia associated with high-dose chemotherapy. METHODS: Unmanipulated growth-factor mobilized PBMC were expanded in media containing daniplestim, leridistim, Promegapoietin, and Progenipoietin (DLPP) and 2% autologous plasma at 4 x 10(5) PBMC/mL, first in 25 cm(2) T-flasks, with sampling on Days 7, 10, 13 and 15, and then in 1264 cm(2) Nunclon Cell Factories, with sampling on Days 7 and 13. RESULTS: In T25-flasks, maximal CFU-GM expansion ([38.2 +/- 9.5]-fold) occurred on Day 10, whereas maximal total cell expansion ([6.7 +/- 1.1]-fold) occurred on Day 15. Production of CD15(+)CD11b(-) and CD15(+)CD11b(+) granulocytic post-progenitors (3.0 +/- 0.4 x 10(6) and 3.7 +/- 0.9 x 10(6), respectively) was also maximal at Day 15. Compared with the previously studied combination of Flt3L, PIXY321, G-CSF, GM-CSF and Epo, the DLPP cocktail performed similarly, with the exception of yielding larger GM colonies at Day 10 and fewer granulocyte post-progenitors on Day 15. In Cell Factories, CFU-GM were expanded (31.6 +/- 14.5)-fold, while total nonadherent cells were expanded (2.6 +/- 0.5)-fold. The two stack Cell Factory cultures seeded with 1.0 x 10(8) unselected PBMC produced approximately 3.3 x 10(6) CFU-GM and 1.3 x 10(8) myeloid post-progenitors. DISCUSSION: Whereas expansion of cell numbers, CFU-GM and granulocytic post-progenitors in Cell Factories mirrored that achieved in T25-flasks, future preclinical studies with the DLPP cytokine combination may be performed in small volumes, with subsequent translation to the larger volume Cell Factories. Sufficient expansion can be achieved using the DLPP cytokine combination in the Cell Factories to provide the numbers of progenitors required for clinical trials.

Low oxygen tension enhances the stimulation and proliferation of human T lymphocytes in the presence of IL-2.

BACKGROUND: Optimization of the culture environment for the ex vivo expansion of T cells is crucial for obtaining the large doses of cells needed for cellular immunotherapy. O2 tension is a key parameter that impacts the proliferation and quality of the expanded T cells. METHODS: Peripheral blood mononuclear cells were stimulated with either PHA or an anti-CD3 monoclonal antibody under 5% (low) or 20% (high) O2 atmospheres. After stimulation, cells were cultured in the presence of IL-2 under either low or high O2 conditions. RESULTS: T cells stimulated and grown under 5% O2 exhibited higher proliferation rates and a mean (n = 11) of 5.8-fold greater total expansion over T cells grown under 20% O2. Stimulation under 5% O2 produced a lasting proliferative effect even after a switch to 20% O2. Examination of apoptosis by the flow cytometry-based TUNEL assay showed a mean (n = 9) of 2.9-fold greater percentage of apoptotic cells under 20% O(2). Flow-cytometric analysis of the IL-2 receptor (CD25) showed that the normal downregulation kinetics - following stimulation-induced CD25 upregulation - were slowed under 5% O(2), such that the 5% O2 cultures had a greater number of CD25+ cells, and those CD25+ cells expressed an average (n = 6) of 41% higher levels of CD25 receptor per cell. No significant O2 tension effects were observed on other surface antigens (CD3, CD28, and CD62L) examined. The key metabolic parameters, specific glucose uptake rate, q(glu), and specific lactate production rate, q(lac), were both increased by a mean (n = 5) of 47% under 5% O2. DISCUSSION: Beyond the physiological significance, improved T-cell proliferation under 5% O2 would allow for decreased culture times in expanding T cells for cellular immunotherapies. Evidence of increased IL-2R expression and reduced apoptosis levels under 5% O2 may help explain this phenomenon.

Characterization of recombinant strains of the Clostridium acetobutylicum butyrate kinase inactivation mutant: need for new phenomenological models for solventogenesis and butanol inhibition?

Two metabolic engineering tools, namely gene inactivation and gene overexpression, were employed to examine the effects of two genetic modifications on the fermentation characteristics of Clostridium acetobutylicum. Inactivation of the butyrate kinase gene (buk) was examined using strain PJC4BK, while the combined effect of buk inactivation and overexpression of the aad gene-encoding the alcohol aldehyde dehydrogense (AAD) used in butanol formation-was examined using strain PJC4BK(pTAAD). The two strains were characterized in controlled pH > or = 5.0 fermentations, and by a recently enhanced method of metabolic flux analysis. Strain PJC4BK was previously genetically characterized, and fermentation experiments at pH > or = 5.5 demonstrated good, but not exceptional, solvent-production capabilities. Here, we show that this strain is a solvent superproducer in pH > or = 5.0 fermentations producing 225 mM (16.7 g/L) of butanol, 76 mM of acetone (4.4 g/L), and 57 mM (2.6 g/L) of ethanol. Strain PJC4BK(pTAAD) produced similar amounts of butanol and acetone but 98 mM (4.5 g/L) of ethanol. Both strains overcame the 180 mM (13 g/L) butanol toxicity limit, without any selection for butanol tolerance. Work with strain PJC4BK(pTAAD) is the first reported use of dual antibiotic selection in C. acetobutylicum. One antibiotic was used for selection of strain PJC4BK while the second antibiotic selected for the pTAAD presence. Overexpression of aad from pTAAD resulted in increased ethanol production but did not increase butanol titers, thus indicating that AAD did not limit butanol production under these fermentation conditions. Metabolic flux analysis showed a decrease in butyrate formation fluxes by up to 75% and an increase in acetate formation fluxes of up to 100% during early growth. The mean specific butanol and ethanol formation fluxes increased significantly in these recombinant strains, up to 300% and 400%, respectively. Onset of solvent production occurred during the exponential-growth phase when the culture optical density was very low and when total and undissociated butyric acid levels were <1 mM. Butyrate levels were low throughout all fermentations, never exceeding 20 mM. Thus, threshold butyrate concentrations are not necessary for solvent production in these stains, suggesting the need for a new phenomenological model to explain solvent formation.

Stoichiometric modeling of Clostridium acetobutylicum fermentations with non-linear constraints.

A stoichiometric model of Clostridium acetobutylicum and related strains has been previously derived. The stoichiometric matrix of the model contains a singularity which has prevented the calculation of a unique set of fluxes which describe the primary metabolic activity. To resolve the singularity, we have developed a non-linear constraint relating the acetate and butyrate uptake fluxes. Subsequently, we developed a software package utilizing a model independent heuristic global optimization approach to solve the resultant non-linear problem. We have validated the use of the non-linear constraint by correlating calculated butyrate production pathway flux profiles with measured intracellular pH profiles. Finally, we examined a controlled batch fermentation to determine that the acid formation pathways play critical roles throughout solventogenesis. The broader usefulness of reformulating the stoichiometric model as a constrained minimization problem is discussed.