Optimization of culture conditions to enhance transfection of human CD34+ cells by electroporation.

The ability to culture CD34+ stem cells, while maintaining their pluripotency, is essential for manipulations such as gene transfection for therapeutic trials. Human peripheral blood (PB) CD34+ cells (> or = 90% purity) were cultured for up to 4 days in serum-free culture medium supplemented with thrombopoietin (TPO), stem cell factor (SCF), Flt-3 ligand (Flt-3L), with or without PIXY321 (IL-3/GM-CSF fusion protein) and human serum. The CD34 mean fluorescence intensity (MFI) and cell cycle status were evaluated daily using flow cytometry and hypotonic propidium iodide. Prior to culture (day 0), 97.0 +/- 0.9%, 1.9 +/- 0.3% and 1.0 +/- 0.6% of the selected CD34+ cells were in G0-G1, S-phase, or G2-M, respectively. After 2-4 days in culture with TPO/SCF/Flt-3L, there was an increase in the percent of cells in S-phase to 26.4 +/- 0.1% without significant loss of CD34 MFI. The addition of PIXY321 increased.the percentage of CD34+ cells in S-phase to 36.3 +/- 4.0%, but the CD34 MFI and numbers of CFU (colony-forming units) were significantly decreased at day 3 when cultured with PIXY321 or various recombinant cytokine combinations that included IL-3 and IL-6. There is an increase from day 0 to day 4 in the percentages of CD34+ with CD38-, HLA-DR-, and c-kit(low), but not Thy-1+ cells. Electroporation with EGFP reporter gene showed that 1-2 days of pre-stimulation in X-VIVO 10 supplemented with TPO/SCF/Flt-3L was necessary and sufficient for efficient transfection. Flow cytometry analysis demonstrated that 22% of the viable cells are CD34+/EGFP+ 48 h post electroporation. The introduced reporter gene appears to be stable as determined by EGFP+/LTC-IC (long-term colony-initiating cells), at 30-40 positive colonies (16 +/- 7%) per 1 x 10(5) electroporated CD34+ cells.

Efficient expression of foreign genes in human CD34(+) hematopoietic precursor cells using electroporation.

Introduction of foreign genes into human CD34(+) hematopoietic precursor cells offers a means to correct inborn errors or to protect human stem cells from chemotherapeutic damage. Electroporation is a non-chemical, nonviral, highly reproducible means to introduce foreign genes into mammalian cells that has been used primarily for rapidly dividing cells. CD34(+) cells isolated from mobilized peripheral blood of patients were cultured for 48 h in serum-free culture medium supplemented with Flt-3 ligand, stem cell factor and thrombopoietin. Cell cycle analysis showed an increase in % S-phase from 2% on day 0 to 28% on day 2 without significant loss of mean fluorescence intensity (MFI). Optimal electroporation conditions for CD34(+) cells were 550 V/cm, 38 ms, 30 microg DNA/500 microl at cell densities between 0.2 x 10(6) and 10 x 10(6) cells/ml resulting in transient EGFP gene expression in 21% (+/- 1%) of CD34(+) precursor cells, as determined by flow cytometry 48 h after electroporation. The more primitive cells were also found to be EGFP(+) as determined by subset analysis using Thy1, CD38, AC133 and c-kit conjugated monoclonal antibodies. Methylcellulose assays on electroporated CD34(+) cells yielded 20% (+/- 7%) EGFP(+) colonies (CFU-GM, BFU-E and CFU-mix) and 22% (+/- 5%) EGFP(+) long-term colony-initiating cells (LTC-IC). The reporter gene was found to be integrated into the LTC-IC genomic DNA as determined by inverse PCR and DNA sequencing. These results suggest that electroporation has the potential to effectively and stably deliver exogenous genes into human hematopoietic precursor cells.

Chromosome 18 breakpoint in t(11;18)(q21;q21) translocation associated with MALT lymphoma is proximal to BCL2 and distal to DCC.

The t(11;18)(q21;q21) translocation has recently been identified as a recurring chromosomal abnormality in a subset of extranodal marginal zone B-cell lymphoma, a low-grade lymphoma of mucosa-associated lymphoid tissue (MALT). Neither the 11q21 nor the 18q21 breakpoints have been characterized by molecular genetic analysis. As a prelude to isolation of the gene(s) involved in this translocation, we have mapped the 18q21 breakpoint region by fluorescence in situ hybridization (FISH) of YAC and PAC clones. We mapped 37 YACs assigned to a 29-cM region within the chromosomal band 18q21. Using nine of these YACs in single- and/or dual-color FISH to analyze three cases of MALT lymphomas with the t(11;18)(q21;q21) translocation, we localized the breakpoints within a 1.6-Mb nonchimeric YAC (938E1). This YAC is useful for the detection of the translocation in metaphase and in interphase cells. A nonchimeric YAC contig of an 8-cM region around the breakpoint comprising nine YACs and a PAC contig of YAC 938E1 were constructed, which enabled the refinement of the breakpoint region in the proximal region of the YAC within a <820-kb segment. This breakpoint is proximal to the BCL2 locus and distal to DCC and DPC4 loci in chromosomal band 18q21.

Costimulatory approaches to adoptive immunotherapy.

Costimulation is critical for induction of full T-cell effector function, and thus represents an attractive immunotherapeutic approach for the treatment of cancer. This review examines these approaches, including ex vivo T-cell expansion, systemic "delivery" of constimulation, tumors transduced or transfected with costimulatory ligands, and vaccine strategies using coimmunization with the genes for costimulatory ligands. Impressive results in animal models have been demonstrated and a wide range of human clinical trials are underway.

The roles of human viruses in the pathogenesis of lymphoma.

There are two families of viruses that contribute to lymphomagenesis in humans: herpesviruses and retroviruses. The two herpesviruses are the Epstein-Barr virus (EBV) and human herpesvirus-8 (HHV-8). EBV is an extremely well-characterized transforming agent: nine viral proteins contribute to transformation in vitro. In contrast, in vivo, the pattern of EBV gene expression varies with different types of malignancies. EBV is associated with endemic Burkitt's lymphoma, acquired immune deficiency syndrome (AIDS)-related lymphoma, post-transplantation lymphoproliferative disease, Hodgkin's disease (HD), and rare T-cell lymphomas. We have summarized studies on the different patterns of viral gene expression and signaling in different EBV-related malignancies, which have begun to reveal how EBV variably contributes to the malignant phenotype in different diseases. HHV-8 is associated with primary effusion lymphomas in patients with AIDS, and the rapidly accumulating information on this virus is summarized. Human T-cell leukemia virus-1 (HTLV-1) is a retrovirus which is the causative agent of adult T-cell leukemia/lymphoma (ATL). The specific mechanism of HTLV-1-mediated T-cell transformation is unclear, but the effects of HTLV-1 on interleukin-2 signaling are reviewed.

Lymphomas in the immunocompromised patient.

Lymphoma is a common opportunistic complication of immunosuppression. Lymphomas in patients with the acquired immunodeficiency syndrome (AIDS) may broadly be divided into four major types: intermediate- or high-grade systemic lymphoma, primary central nervous system (CNS) lymphoma, Hodgkin's disease (HD) and primary effusion lymphoma. Multiple active regimens have been identified for patients with AIDS-related systemic lymphoma. However, despite high initial complete response rates, most studies have reported a median survival of less than 1 year for these patients, with approximately half of the patients dying from lymphoma and half from opportunistic infections or other AIDS-related complications. The standard therapeutic approach for patients with AIDS-related primary CNS lymphoma is radiotherapy, although recent studies using combinations of chemotherapy with radiotherapy may offer an improvement in therapy for this group of patients who have very poor overall prognosis. Lymphoproliferative disease in patients after solid organ or bone marrow transplantation represents with a spectrum of disorders. No standard approach for therapy in this group of patients has been clearly established.