No abstract

Hematopoietic stem cells are widely recognized as attractconditions employed, Ͼ75% of the target cells retained the ive targets for gene therapy but current protocols to trans-CD34 + Lin − primitive phenotype after 4-5 days in culture; of duce these cells using recombinant retroviral vectors are those у25% expressed a high level of GFP detectable by inefficient. To evaluate optimization of retroviral transducboth flow cytometric analysis and fluorescence tion of hematopoietic stem cells and stability of gene microscopy. When transduced cells were cultured in clonexpression in their progeny, the green fluorescent protein ogenic progenitor assays, GFP fluorescence was readily (GFP) was explored as a reporter. We first improved sensidetected in situ, indicating that GFP expression was stable tivity of detection Ͼ100-fold over that achieved previously and not detrimental to the differentiative potential of the by using a novel retroviral vector (termed MGIN) expresstransduced CD34 + Lin − cells. We conclude that GFP is ing a high level of an enhanced GFP gene. Primitive effective as a vital marker to quantify retrovirus-mediated human hematopoietic cells bearing the CD34 surface gene transfer into human hematopoietic and perhaps antigen and lacking lineage differentiation markers other types of stem/progenitor cells, and monitor (CD34 + Lin − ) were transduced with the MGIN vector using gene expression during their subsequent cell lineage a clinically applicable supernatant procedure. Under the determinations.Keywords: gene therapy; gene transfer; gene expression; retroviral vectors; GFP; hematopoietic stem cells HSPC, the second problem associated with retroviral vecIntroduction tor-based gene therapy is low levels of sustained gene Hematopoietic stem and progenitor cells (HSPC) provide expression. Retroviral vectors derived from Moloney an attractive target for gene therapy because they have murine leukemia virus (MoMLV) are the most commonly the potential to continue producing progeny cells conused retroviral vectors in clinical trials. In a standard containing a therapeutic gene indefinitely. Hematological figuration, the gene of interest is placed under the trandiseases potentially benefiting from HSPC-based gene scriptional control of the viral long terminal repeat (LTR) therapy approaches include hereditary hemoglobinosince gene expression driven by LTR is generally higher pathies, immune deficiencies and disorders of phagocytic than by an internal promoter. 9,10 However, it has been cells, as well as other diseases such as acquired immunoreported that MLV LTR-mediated gene expression is fredeficiency syndrome and cancer. 1 Retroviral vectors, quently down-regulated during differentiation of which are being used in the majority of current clinical HSPC. 11,12 Because the LTR of the murine stem cell virus trials, are a primary choice as the vehicle for gene deliv-(MSCV) retroviral vector is permissive for expression in ery since they are capable of integrating into cellular murine HSPC, 8,13 we were interested...

Detection of rare, circulating tumor cells (CTC's) in human peripheral blood is a potential indicator of prognosis and diagnosis in oncology. Typical methods to detect these CTC's are either by immunocytochemistry (ICCS) or RT-PCR. However without accurate, rapid, and reproducible enrichment processes, these detection techniques are labor intensive and/or unreliable. In this article, a repeatable enrichment process that included a flow-through immunomagnetic cell separation system, the quadrupole magnetic sorter (QMS) was optimized with the aid of a statistical analysis software package. The QMS was operated in a negative mode of operation by immunomagnetically targeting normal human peripheral blood lymphocytes (PBL) through the CD45 surface marker. Three head and neck squamous carcinoma cell lines (HNSCC), Detroit-562, SCC-4, and CAL-27, were used to determine the sensitivity of RT-PCR for the epidermal growth factor receptor (EGFR) in spiked PBL. The detection purity needed for detection was found to be one cell in 10 4 , one cell in 10 3 , and one cell in 10 5 for the Detroit-562, SCC-4, and CAL-27, respectively. The actual number of cancer cells needed for RT-PCR detection ranged from 30 to 1 cell. To mimic the potential concentration of rare CTC present in peripheral blood of cancer patients, the spiking concentration was chosen to be one cancer cell per 10 5 total leukocytes from healthy donors. Using a single step immunomagnetic labeling, the final, optimized enrichment process produced a 57.6 6 30.3-fold (n 5 6) enrichment of the rare cancer cells with a final cancer cell recovery of (77.8 6 6.6)%. q 2007 Clinical Cytometry Society Key terms: immunomagnetic cell separation; circulating tumor cells; reverse transcriptase polymerase chain reaction; head and neck squamous carcinoma cell; Detroit-562; epidermal growth factor receptor; immunocytochemistry staining; quadrupole magnetic sorter Head and Neck cancer comprises *6% of all cancers which results, globally, in *550,000 cases a year. According to the statistics of American Cancer Society, 39,250 new cases of cancer of the oral cavity, oropharynx, pharynx, and larynx are estimated to occur in the US in 2005, and 10,090 deaths were estimated to occur. Greater than 90% of these cases of head and neck cancer are classified as squamous cell carcinomas. For all stages combined, the 5-year survival rate is approximately 58.7%, and this rate has increased slowly in the last 20 years. About 40-50% of the patients will later develop locoregional recurrences, and approximately 80% of these recurrences occur within the first 2 years.Both recent and historical studies have shown that tumor cells are likely to exist in the bone marrow or in the peripheral blood of various types of cancer patients (1-6). It has been presumed that those tumor cells shed from the original tumors and are considered to contribute to the

A novel magnetic support was prepared by an oxidization-precipitation method with poly(vinyl alcohol) (PVA) as the entrapment material. Transmission electron microscopy indicated that the magnetic particles had a core-shell structure, containing many nanometer-sized magnetic cores stabilized by the cross-linked PVA. The particles showed a high magnetic responsiveness in magnetic field, and no aggregation of the particles was observed after the particles had been treated in the magnetic field. These facts indicated that the particles were superparamagnetic. Cibacron blue 3GA (CB) was coupled to the particles to prepare a magnetic affinity support (MAS) for protein adsorption. Lysozyme was used as a model protein to test the adsorption properties of the MAS. The adsorption equilibrium of lysozyme to the MAS was described by the Langmuir-type isotherm. The capacity for lysozyme adsorption was more than 70 mg/g MAS (wet weight) at a relatively low CB coupling density (3-5 micromol/g). In addition, 1.0 M NaCl solution could be used to dissociate the adsorbed lysozyme. Finally, the MAS was recycled for the purification of alcohol dehydrogenase (ADH) from clarified yeast homogenates. Under proper conditions, the magnetic separation yielded over 5-fold purification of the enzyme with 60% recovery of the enzyme activity.

Efficient conversion of carbon dioxide is of great interests to today's endeavors in controlling greenhouse gas emission. A multienzyme catalytic system that uses carbon dioxide and ethanol to produce L-lactate was demonstrated in this work, thereby providing a novel reaction route to convert bio-based ethanol to an important building block for synthesis biodegradable polymers. The synthetic route has a unique internal cofactor regeneration cycle, eliminating the need of additional chemical or energy for cofactor regeneration. Lactate was successfully synthesized with 41% of ethanol converted in a batch reaction, while a turnover number of 2.2 day⁻¹ was reached for cofactor regeneration in a reaction with continuous feeding of ethanol. A kinetic model developed based on reaction kinetic parameters determined separately for each reaction step predicted well the reaction rates and yields of the multienzyme reaction system.