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E T Papoutsakis

Publications and source records attributed to E T Papoutsakis.

At least 55 records · Page 3Linked to original sources
PubMed1996

Hematopoietic cell culture therapies (Part II): Clinical aspects and applications.

High-dose chemotherapy, followed by hematopoietic stem cell transplantation, holds significant promise for increasing the probability of long-term remission and possibly cure in a variety of cancers. Hematopoietic cell culture, or ex vivo expansion of hematopoietic cells, may play a significant role in reducing the danger and expense associated with the transplantation procedure. Phase I clinical trials have shown that ex vivo expanded cells have no significant toxicities, and some benefits. Ex vivo expansion of hematopoietic cells is likely to find other applications in gene therapy, tumor purging, production of dendritic cells for immunotherapy and the production of mature blood cells for transfusion therapies.

Antigens, CD34↗
PubMed1996

Ex vivo culture systems for hematopoietic cells.

During the past few years, hematopoietic cell culture technologies for transplantation therapies have progressed significantly on several fronts. Advances include the discoveries of the growth factors thrombopoietin and Flt-3 ligand, the development of a variety of bioreactor systems, and results from preliminary clinical trials that demonstrate the efficacy of ex vivo expanded hematopoietic cells for transplantation therapy.

Blood↗
PubMed1996

Comparison of whole serum-deprived media for ex vivo expansion of hematopoietic progenitor cells from cord blood and mobilized peripheral blood mononuclear cells.

A whole serum-deprived (WSD) medium was developed and optimized for expansion of colony-forming cells (CFC) in cord blood (CB) mononuclear cell (MNC) cultures. This medium was compared with four commercially available WSD media (commercial media), three WSD media whose compositions have been publicly disclosed (public media), two serum-containing media, and two basal media, for cell and CFC expansion in 10-day CB and mobilized peripheral blood (PB) MNC cultures supplemented with interleukin-3 (IL-3), IL-6, and stem cell factor (SCF). Selected WSD media and both serum-containing media gave significant CFC expansion in CBMNC and PBMNC cultures. The serum-containing human long-term medium gave the greatest cell (3.0-fold) and CFC (25-fold) expansions in CBMNC cultures, whereas our medium maintained the most cells (93% of input) and gave the greatest CFC expansion (6.1-fold) for PBMNC cultures. Of the commercial media, Progenitor-34 gave the greatest cell expansion (1.2-fold) and X VIVO-10 gave the greatest CFC expansion (11-fold) for CBMNC cultures, and Progenitor-34 maintained the most cells (83% of input) and gave the greatest CFC expansion (3.1-fold) for PBMNC cultures. Of the public media (including ours), our medium gave the greatest cell (1.4-fold) and CFC (6.1-fold) expansion for CBMNC cultures. Although there were slight correlations between cell and CFC expansion in 10-day CBMNC and PBMNC cultures (r2 of 0.848 and 0.594, respectively), the correlations did not give reliable predictions for medium selection. In addition, the different media favored expansion of different CFC types and performed differently for cultures using different cell sources (CB versus PB). Taken together, these results suggest that media must be carefully screened for the cell source to be cultured and the cell type(s) to be produced (e.g. total cells, CFC).

Cell Division↗
PubMed1996

Genetic manipulation of acid formation pathways by gene inactivation in Clostridium acetobutylicum ATCC 824.

Integrational plasmid technology has been used to disrupt metabolic pathways leading to acetate and butyrate formation in Clostridium acetobutylicum ATCC 824. Non-replicative plasmid constructs, containing either clostridial phosphotransacetylase (pta) or butyrate kinase (buk) gene fragments, were integrated into homologous regions on the chromosome. Integration was assumed to occur by a Campbell-like mechanism, inactivating either pta or buk. Inactivation of the pta gene reduced phosphotransacetylase and acetate kinase activity and significantly decreased acetate production. Inactivation of the buk gene reduced butyrate kinase activity, significantly decreased butyrate production and increased butanol production.

Acetates↗
PubMed1995

Effects of CD34+ cell selection and perfusion on ex vivo expansion of peripheral blood mononuclear cells.

Ex vivo expansion of peripheral blood mononuclear cells (MNCs), cultured both directly and after selection for CD34+ cells, was compared in static and continuously perfused cultures containing interleukin (IL)-3, IL-6, granulocyte colony-stimulating factor (G-CSF), and stem cell factor (SCF). Cultures inoculated with either MNCs or CD34+ cells produced cells that were remarkably similar after 10 days of culture, as evidence by cell morphology, expression of CD34, CD33, CD15, and CD11b, and the fractions of cells giving rise to colony-forming units granulocyte-monocyte (CFU-GM) and long-term culture-initiating cells (LTC-IC). Static and perfusion cultures gave similar average total cells and CFU-GM expansions for both MNC and CD34+ cell cultures. However, those samples that performed poorly in static culture performed at near-normal levels in perfusion. In addition, perfusion supported higher LTC-IC numbers for both MNC and CD34+ cell cultures. While total cell expansion was about ten times greater in CD34+ cell cultures (approximately 100-fold), CFU-GM expansion (approximately 20-fold) was similar for both MNC and CD34+ cell cultures. The similar distribution of cell types produced in MNC and CD34+ cell cultures allows direct comparison of total and colony-forming cell production. After 15 days in perfusion, MNC cultures produced 1.5-, 2.6-, and 2.1-fold more total cells, CFU-GM, and LTC-IC, respectively, than the same sample selected and cultured as CD34+ cells. Even if the CD34+ selection process was 100% efficient, CFU-GM production would be 1.5-fold greater for MNCs than for CD34+ cells.

Antigens, CD↗
PubMed1995

Cell adhesion in animal cell culture: physiological and fluid-mechanical implications.

We have reviewed the general forces through which cells interact with substrata in their first nonspecific contact. The complex, fast-emerging biology of specific cell adhesion and the structure of the extracellular matrix were reviewed in substantial detail, and the most updated conceptual model of biological cell adhesion was assembled from past efforts and new literature data. The chemistries of the various possible substrata for cell adhesion have been reviewed extensively in the past, and here only a brief summary was presented, with particular emphasis on the materials for traditional and porous microcarriers. The fascinating molecular and cellular implications of cell adhesion were reviewed in detail to establish that cell adhesion and the extracellular matrix provide more than structural support for the cells and their assemblies, and that in fact they constitute fundamental regulators of cell function, metabolism, and differentiation. We reviewed the fluid-mechanical mechanisms of cell damage in microcarrier systems and provided experimental evidence for the importance of the cell-adhesion quality in the ability of cells to withstand fluid forces in bioreactors. We provided evidence that the interplay of cell adhesion and fluid forces is likely to produce cell responses more complex than that of simple life and death, and we suggested that such responses are awaiting investigation and exploration for new applications and culturing possibilities. We also reviewed the experimental evidence on the importance of cell adhesion in cell and microcarrier aggregation and discussed the implications of such aggregation on the culturing environment and the operation of bioreactors. Finally, we discussed the possible implications of cell adhesion as it relates to the developing field of tissue engineering, using the example of bone marrow culture, which involves a large variety of cells and constitutes one of the most complex cell culture systems.

Animals↗
PubMed1994

Cell aggregation in a Chinese hamster ovary cell microcarrier culture affects the expression rate and N-linked glycosylation of recombinant mouse placental lactogen-1.

A microcarrier culture of Chinese hamster ovary (CHO) cells, expressing the N-glycosylated recombinant protein mouse placental lactogen I (mPL-I), was found to form large cellular aggregates (400 to 600 microns in diameter). There was increased accumulation of lower molecular sized mPL-I glycoforms in cultures containing the large cellular aggregates at pH 7.3, but not at pH 7.6. Specific rates of mPL-I expression were found in the cultures with cellular aggregates at both pH values (7.3 and 7.6). These findings are interpreted in the light of our earlier studies that showed that extracellular pH and elevated ammonia concentrations affect both the glycosylation and the expression rates of mouse placental lactogen I.

Ammonia↗
PubMed1994

Expression of plasmid-encoded aad in Clostridium acetobutylicum M5 restores vigorous butanol production.

Mutant M5 of Clostridium acetobutylicum ATCC 824, which produces neither butanol nor acetone and is deficient in butyraldehyde dehydrogenase (BYDH), acetoacetate decarboxylase, and acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase activities, was transformed with plasmid pCAAD, which carries the gene aad (R. V. Nair, G. N. Bennett, and E. T. Papoutsakis, J. Bacteriol, 176:871-885, 1994). In batch fermentation studies, aad expression restored butanol formation (84 mM) in mutant M5 without any acetone formation or any significant increase in ethanol production. The corresponding protein (AAD) appeared as a ca. 96-kDa band in a denaturing protein gel. Expression of AAD in M5 resulted in restoration of BYDH activity and small increases in the activities of acetaldehyde dehydrogenase, butanol dehydrogenase, and ethanol dehydrogenase. These findings suggest that BYDH activity in C. acetobutylicum ATCC 824 resides largely in AAD, and that AAD's primary role is in the formation of butanol rather than of ethanol.

Alcohol Oxidoreductases↗
PubMed1994

Molecular characterization of an aldehyde/alcohol dehydrogenase gene from Clostridium acetobutylicum ATCC 824.

A gene (aad) coding for an aldehyde/alcohol dehydrogenase (AAD) was identified immediately upstream of the previously cloned ctfA (J. W. Cary, D. J. Petersen, E. T. Papoutsakis, and G. N. Bennett, Appl. Environ. Microbiol. 56:1576-1583, 1990) of Clostridium acetobutylicum ATCC 824 and sequenced. The 2,619-bp aad codes for a 96,517-Da protein. Primer extension analysis identified two transcriptional start sites 83 and 243 bp upstream of the aad start codon. The N-terminal section of AAD shows homology to aldehyde dehydrogenases of bacterial, fungal, mammalian, and plant origin, while the C-terminal section shows homology to alcohol dehydrogenases of bacterial (which includes three clostridial alcohol dehydrogenases) and yeast origin. AAD exhibits considerable amino acid homology (56% identity) over its entire sequence to the trifunctional protein encoded by adhE from Escherichia coli. Expression of aad from a plasmid in C. acetobutylicum showed that AAD, which appears as a approximately 96-kDa band in denaturing protein gels, provides elevated activities of NADH-dependent butanol dehydrogenase, NAD-dependent acetaldehyde dehydrogenase and butyraldehyde dehydrogenase, and a small increase in NADH-dependent ethanol dehydrogenase. A 957-bp open reading frame that could potentially encode a 36,704-Da protein was identified upstream of aad.

Alcohol Dehydrogenase↗
PubMed1993

Sequence and arrangement of two genes of the butyrate-synthesis pathway of Clostridium acetobutylicum ATCC 824.

The genes encoding both Clostridium acetobutylicum ATCC 824 butyrate synthesis pathway enzymes, phosphotransbutyrylase (ptb) and butyrate kinase (buk), were sequenced. The genes are immediately adjacent on the chromosome, with ptb preceding buk. A single transcription start point (tsp) was identified 57 bp upstream from the ptb start codon by primer extension analysis. The ptb and buk genes appear to form an operon. A putative Rho-independent terminator structure was identified 26 bp downstream from buk.

Amino Acid Sequence↗
PubMed1993

Determination of plasmid copy number and stability in Clostridium acetobutylicum ATCC 824.

The copy number and stability of several plasmid vectors in Clostridium acetobutylicum ATCC 824 were determined. The protocols were modified from the traditional ones to overcome the problems associated with unusual behavior of C. acetobutylicum cells on solid medium. The plasmid copy numbers of pSYL2, pFNK1, pFNK3, and pFNK5 in strain ATCC 824 were 14, 8, 6, and 6, respectively. pSYL2 and pFNK1 were segregationally stable, since the fractions of plasmid-carrying cells after 60 generations of growth without antibiotic (erythromycin) were 73% and 77%, respectively. Vector pFNK1 carrying fermentative genes was found to be rather unstable. The observed instability seemed to be due to the complex host-plasmid interactions by amplified expression of enzymes involved in the tightly regulated primary metabolism of C. acetobutylicum.

Clostridium↗
PubMed1993

Expansion of primitive human hematopoietic progenitors in a perfusion bioreactor system with IL-3, IL-6, and stem cell factor.

Present methods for long-term hematopoietic culture (LTHC) employ a static culture environment which is not well-characterized. Primitive long-term culture-initiating cell (LTC-IC) numbers have been shown to decline in conventional static human LTHC, even with exogenous cytokine combinations. We have expanded human hematopoietic cells from umbilical cord blood on a preformed marrow stroma with synergistic cytokine combinations in a novel perfusion bioreactor system, which continuously maintained culture conditions within desired ranges. Interleukin-3 (IL-3) and interleukin-6 (IL-6) in perfusion culture resulted in rapid 7-day expansion of granulocyte-macrophage colony forming units (CFU-GM, 11-fold), erythroid burst-forming units (BFU-E, 2.5-fold), and granulocyte-erythroid-macrophage colony forming units (CFU-Mix, 2.4-fold), compared to 6-fold, 1.4-fold, and no expansion, respectively, in static cultures. Addition of stem cell factor (SCF) to IL-3/IL-6 in static culture increased the extent of CFU-GM expansion (to 9-fold), but did not result in BFU-E or CFU-Mix expansion. In perfusion cultures with IL-3/IL-6/SCF, much greater expansions of CFU-GM (18-fold) and CFU-Mix (5.3-fold) were obtained. More importantly, expansion of LTC-IC (nearly 3-fold in two of three experiments) was only obtained with IL-3/IL-6/SCF and perfusion. The ability to expand hematopoietic cells while maintaining or expanding primitive progenitors has potential clinical applications in bone marrow transplantation and gene therapy.

Cell Count↗
PubMed1993

Culture pH affects expression rates and glycosylation of recombinant mouse placental lactogen proteins by Chinese hamster ovary (CHO) cells.

Glycosylation patterns and specific expression rates of the recombinant protein mouse placental lactogen-I (mPL-I) by Chinese hamster ovary (CHO) cells varied significantly over the extracellular pH (pHe) range of 6.1 to 8.7. The maximum specific mPL-I expression rates occurred between pHe 7.6 and 8.0. The pHe effect on protein expression was confirmed using a different CHO cell expressing the unglycosylated recombinant protein mouse placental lactogen-II (mPL-II). Decreases in the extent of glycosylation of mPL-I were observed at low (below 6.9) and high (above 8.2) pHe values. The pHe dependent variations in mPL-I accumulation in the supernatant as well as in glycosylation patterns were not the result of enzymatic degradation in the culture medium.

Animals↗
PubMed1993

In vivo methylation in Escherichia coli by the Bacillus subtilis phage phi 3T I methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824.

The restriction endonuclease Cac824I has been shown to be a major barrier to electrotransformation of Clostridium acetobutylicum ATCC 824 (L. D. Mermelstein, N. E. Welker, G. N. Bennett, and E. T. Papoutsakis, Bio/Technology 10:190-195, 1992). Methylation by the phi 3T I methyltransferase encoded by Bacillus subtilis phage phi 3T was shown to protect plasmid DNA from restriction by Cac824I. Expression in Escherichia coli of the phi 3tI gene (which encodes the phi 3T I methyltransferase) from pAN1, which replicates via the p15A origin of replication, was sufficient to completely methylate coresident E. coli-C. acetobutylicum shuttle vectors with ColE1 origins of replication. Three shuttle vectors (pIMP1, pSYL2, and pSYL7) methylated in this manner were used to efficiently electrotransform strain ATCC 824. These vectors could not be introduced into strain ATCC 824 when unmethylated because the E. coli portions of the plasmids contain a large number of Cac824I sites. This method obviates the need to use B. subtilis-C. acetobutylicum shuttle vectors with few Cac824I sites to introduce DNA into C. acetobutylicum ATCC 824.

Amino Acid Sequence↗
PubMed1992

Vector construction, transformation, and gene amplification in Clostridium acetobutylicum ATCC 824.

In order to alter the primary metabolism of C. acetobutylicum, we have constructed E. coli- or B. subtilis-C. acetobutylicum shuttle vectors that could be used to deliver homologous fermentative genes into C. acetobutylicum ATCC 824. The plasmid copy number and plasmid stability in C. acetobutylicum for several of these plasmids were determined. We have also developed a protocol for the electrotransformation of C. acetobutylicum ATCC 824. Difficulty in the transformation of C. acetobutylicum ATCC 824 with vectors containing DNA from E. coli plasmids was found to be due to the existence of a restriction system in this strain. This type II restriction endonuclease, named Cac824I, recognizes the sequence 5'-GCNGC-3' and cuts ColE1 plasmids frequently. One of the vectors, pFNK1, possessing a variety of unique cloning sites was used in the amplification of one acid (PTB) and one solvent (AADC) formation gene. The corresponding enzyme activities were amplified in C. acetobutylicum as shown by enzyme assays and SDS-PAGE gels of cell extracts.

Bacillus subtilis↗