Generation and selection of immunized Fab phage display library against human B cell lymphoma

The approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production ofmonoclonal antibodies by the traditional hybridoma technology is costly, and the resulting murine antibodies often have the disadvantage of triggering human anti-mouse antibody （HAMA） response. Therefore recombinant Fab antibodies generated by the phage display technology can be a suitable alternative in managing B cell lymphoma. In this study, we extracted total RNA from spleen cells of BALB/c mice immunized with human B lymphoma cells, and used RT-PCR to amplify cDNAs coding for the κ light chains and Fd fragments of heavy chains. After appropriate restriction digests, these cDNA fragments were successively inserted into the phagemid vector pComb3H-SS to construct an immunized Fab phage display library. The diversity of the constructed library was approximately 1.94× 10^7. Following five rounds of biopanning, soluble Fab antibodies were produced from positive clones identified by ELISA. From eight positive clones, FabC06, FabC21, FabC43 and FabC59 were selected for sequence analysis. At the level of amino acid sequences, the variable heavy domains （VH） and variable light domains （VL） were found to share 88-92% and 89-94% homology with sequences coded by the corresponding murine germline genes respectively. Furthermore, reactivity with membrane proteins of the B cell lymphoma was demonstrated by immunohistochemistry and western blotting. These immunized Fab antibodies may provide a valuable tool for further study of B cell lymphoma and could also contribute to the improvement of disease therapy.

Abnormal expression of PACAP gene in patients with myelodysplastic syndrome

Objective： To explore the expression pattern of PACAP gene in patients with myelodysplastic syndrome （MDS）. Methods： The expression pattern of PACAP gene in CD34＋ cells from MDS patients was determined by microarray, confirmed in expanding samples by quantitative real-time PCR in bone marrow mononuolear cells. Results： The transcription of PACAP gene was found significantly down-regulated in patients with MDS in comparison with that in control samples, with a means of 220.1 in MDS-RA and 116.8 in MDS-RAEB （P 〈 0.05 ）. Conclusion： PACAP gene is expressed significantly lower in patients with MDS.

Direct contact with bone marrow stromal cells promotes the invasions of SHI-1 leukemia cells

Background Interactions of tumor cells with the microenvironment were deemed to promote the tumor invasion and metastasis. CXC chemokine receptor 4 （CXCR4） and extracellular matrix metalloproteinase inducer （EMMPRIN） had reported to participate in this process. However the roles of bone marrow microenvironment in leukemic infiltration were not well investigated. Methods A co-culture system between SHI-1 cells and bone marrow stromal cells （BMSCs） is used to simulate the interactions of leukemic cells with their microenvironment. The trans-matrigel invasion was used to detect the capability of SHI-1 cells invasion. The BMSCs and SHI-1 cells were mixed in a ratio of 1：10 and added to the millicell chamber coated with matrigel. Either the co-culture supernatant or the functional blocking peptide of CXCR4 and EMMPRIN were added to the trans-matrigel invasion system. The expressions of EMMPRIN in SHI-1 cells and BMSCs were detected by RT-PCR. The changes of the expression of matrix metalloproteinase-2, 9 （MMP-2, MMP-9）, tissue inhibitor of metalloproteinase 2 （TIMP-2）, and CXCR4 mRNA in SHI-1 cells were determined by real-time PCR. The concentration of stromal cell derived factor 1 （SDF-1） in serum free supernatant was measured by ELISA. Results Both SHI-1 cells and BMSCs express EMMPRIN. SHI-1 cells could hardly invade the matrigel membrane; the co- culture supernatant did not induce the invasion of SHI-1 cells. When contacting directly with BMSCs, SHI-1 cells invaded to the lower chamber of millicell were significantly increased. The functional blocking peptide of CXCR4 and EMMPRIN could significantly inhibit the invasion triggered by BMSCs. When co-culturing with BMSCs, the expression of CXCR4, MMP-2, MMP-9 and TIMP-2 mRNA in SHI-1 cells were significantly elevated in company with a significantly higher level of SDF-1 in the co-cultured serum-free supernatant. Conclusion The interactions of leukemic cells and BMSCs play important roles in leukemic cell infiltration.

Clinical,cytogenetic and dual-color FISH studies on five cases of myelodysplastic syndrome or acute myeloid leukemia patients with 1;7 translo cation

To study the clinical and cytogenetic characteristics of four patients with myel odysplastic syndrome (MDS) and one with acute myeloid leukemia experiencing t(1; 7) Methods Five patients seen in our hospital from 1992 to 2001 were diagnosed as MDS and acute myelocytic leukemia (AML) according to the French American British (FAB) criteria Chromosomes were prepared using the direct method as well as 24 hou r unstimulated cultures of fresh heparinized bone marrow for each subject, while R banding was used to analyze karyotypes Dual color fluorescence in situ hy bridization (FISH) using SpectrumRed and SpectrumGreen directly labeled chromoso me 1 specific α satellite DNA probe (red) and chromosome 7 specific α sat ellite DNA probe (green) was performed for three cases Results Of the five patients, three had 1;7 translocation due to a long history of expos ure to benzene In three cases, dual color FISH resulted in three red signals and two green ones, in which one red signal adjoining one green signal in 27 6% , 84% and 18 5% metaphases, respectively Conclusions Exposure to benzene may be the cause for Chinese MDS and AML patients with t(1;7 ) translocation The result of dual color FISH convincingly confirmed that the centromere of the derivative chromosome 7p/1q resulting from 1;7 translocation was made up of centromeres from both chromosomes 1 and 7