Marcia M. Bieber

Detection of fetal trisomies 21 and 18 from maternal blood using triple gradient and magnetic cell sorting

PROBLEM: The need for an inexpensive and reproducible technique for noninvasive prenatal diagnosis by fetal cell isolation from maternal blood.

Rapid cytotoxicity of human B lymphocytes induced by VH4-34 (VH4.21) gene-encoded monoclonal antibodies, II

We have previously described two human cold agglutinin MoAbs 216 and A6(H4C5), that are derived from the VH4‐34 (VH4.21) gene that bind specifically to a cell surface ligand on human B lymphocytes. In this study, we report that binding of 216 and A6(H4C5) leads to rapid killing of target B cells. This complement‐independent cytotoxicity was measured by three independent assays, cell viability dye uptake on FACS, 3H‐thymidine uptake, and the 3(4,5)‐dimethylthiazol‐2,5‐diphenyl tetrazolium bromide (MTT) assay. Cytotoxicity was specific for CD20+ mononuclear cells in human spleen and peripheral blood. The MoAbs were also cytotoxic to human B cell lines Nalm‐6, OCI‐LY8, Arent and SUP‐B8, but not to T cell lines HuT 78 and PEER. As observed by scanning electron microscopy, membrane pores were formed within 15 min of exposure to the MoAbs. Cytotoxic activity was dependent on MoAb concentration and temperature of exposure. Killing was greater at 4°C than 37°C. Sodium azide and EDTA did not block the cytotoxic activity. No DNA fragmentation typical of apoptosis was observed. This rapid cytotoxic activity, independent of physiologic cellular processes and independent of complement, suggests a novel mechanism of cell death via membrane perturbations.

One-step enrichment of nucleated red blood cells: A potential application in perinatal diagnosis

We describe a discontinuous triple density gradient to obtain a 25-fold enrichment of nucleated red blood cells from mononuclear cells, granulocytes, and mature red blood cells in a single centrifugation step.

Hedgehog signaling regulates drug sensitivity by targeting ABC transporters ABCB1 and ABCG2 in epithelial ovarian cancer.

A major challenge of successful chemotherapy in ovarian cancer is overcoming intrinsic or acquired multi‐drug resistance caused by active drug efflux mediated by ATP‐binding cassette (ABC) transporters. Regulation of these transporters in ovarian cancer is poorly understood. We have found that abnormal expression of the hedgehog (Hh) signaling pathway transcription factor Gli1 is involved in the regulation of ABC transporters ABCB1 and ABCG2 in ovarian cancer. Hh is a known regulator of cancer cell proliferation and differentiation in several other types of invasive and metastatic malignancies. Our work has demonstrated that Gli1 is abnormally activated in a portion of ovarian cancers. Inhibition of Gli1 expression decreases ABCB1 and ABCG2 gene expression levels and enhances the response of ovarian cancer cells to certain chemotherapeutic drugs. The underlying mechanism is a direct association of Gli1 with a specific consensus sequence located in the promoter region of ABCB1 and ABCG2 genes. This study provides new understanding of ABC gene regulation by Hh signaling pathway, which may lead to the identification of new markers to detect and to anticipate ovarian cancer chemotherapy drug sensitivity.

Human CD5+ B lymphocytes (B-1 cells) decrease in peripheral blood during pregnancy

Pregnancy is a unique immunologic state where a natural homeostasis exists between antigenically different tissues. Several earlier studies have addressed the fluctuations in the number and/or function of lymphocytes, including B cells during pregnancy, but changes within the subsets of B lymphocytes, conventional (CD5-) and B-1 (CD5+), have not been addressed. Here we demonstrate that the frequency of B-1 cells decreases dramatically during pregnancy, whereas the frequency of conventional B cells remains relatively constant. The missing B-1 cells return to pre-pregnancy levels 8-10 weeks after parturition. The polyreactive autoantibodies secreted by B-1 cells have been implicated in autoimmunity and immune regulation. The possible role of B-1 cells during pregnancy will be discussed in that context.

Wnt and Hedgehog Gene Pathway Expression in Serous Ovarian Cancer

Objective: Ovarian cancer has very heterogeneous histological classification, and response to therapy of the same grade and type varies. We studied genes in the Wnt and hedgehog (Hh) pathways, which are essential for embryonic development and which play critical roles in proliferation in a variety of human cancers. Variations in these pathway genes causing proliferation could play a role in the variation in tumor progression and response to therapy. Methods/Materials: Using real-time polymerase chain reaction, we studied 16 primary grade 3 International Federation of Gynecology and Obstetrics stage III serous ovarian cancer samples for expression of the Wnt pathway gene AXIN2, fibroblast growth factor 9, and Hh pathway gene expressions of glioma-associated oncogene 1, glioma-associated oncogene 2, patched homolog 1, patched homolog 2, Indian Hedgehog (HH), sonic HH, and Smoothened, a G protein-coupled receptor protein. Normal ovary epithelial cell line was used as control. Results: We found wide variation of up-regulation of pathway component and target genes in the primary tumor samples and apparent cross talk between the pathways. AXIN2, a Wnt target gene, showed increased expression in all serous ovarian cancer samples. Fibroblast growth factor 9 was also overexpressed in all tumors with greater than1000-fold increase in gene expression in 4 tumors. Expression of Hh pathway genes varied greatly. More than half of the tumor samples showed involvement of Hh signaling or pathway activation either by expression of transcription factors and Hh ligands or by overexpression of Indian HH/sonic HH and the receptor-encoding patched homolog 1/patched homolog 2. Conclusion: We found a wide variation in fold expression of genes involved in the Wnt and Hh pathway between patient samples.

Biology and virology of the human malignant lymphomas. 1st Milford D. Schulz lecture

Permanent cell lines have been established from twelve diffuse histiocytic lymphomas (SU‐DHL‐1 to ‐12), three American Burkitts lymphomas (SU‐AmB‐1 to ‐3), two acute lymphoblastic leukemias (SU‐ALL‐1 and ‐2), and three diffuse undifferentiated lymphomas (SU‐DUL‐1, ‐2, and ‐3). The cultured cells displayed neoplastic characteristics, as manifested by hetero‐transplantability in congenitally athymic nude mice and by the presence of cytogenetic abnormalities in early passage generations. Functional and marker studies revealed that the three American Burkitts lymphomas, as well as several of the diffuse histiocytic and undifferentiated lymphomas, were of B‐lymphocytic origin, whereas the two acute lymphoblastic leukemias were both of T‐lymphocytic origin. Two of the cell lines, SU‐DHL‐1 and ‐2, appeared to be of true histiocytic origin; two others exhibited no markers and were designated as “null” cells. All ten of the DHL cell lines studied to date, as well as SU‐DUL‐1, have been devoid of Epstein‐Barr virus (EBV) genomes by the EBNA test, whereas two of the three American Burkitts lymphoma cell lines were positive. Spontaneous production of a C‐type RNA virus was first detected in post‐mitochondrial cytoplasmic fractions and culture fluids of the SU‐DHL‐1 cell line. Screening assays for the detection of reverse transcriptase‐positive particles in the culture fluids of the other cell lines indicate that eight of the fifteen cell lines tested to date have spontaneously initiated C‐type RNA virus production. After partial purification by ion‐exchange and affinity chromatography, the reverse transcriptase of the virus isolated from SU‐DHL‐1 cells is partially inhibited by antibodies to the reverse transcriptases of C‐type viruses of subhuman primate and endogenous feline, but not of murine, origin. Conversely, antibody prepared against the purified SU‐DHL‐1 viral reverse transcriptase, at concentrations which maximally inhibit the homologous enzyme, partially inhibits the reverse transcriptases of subhuman primate C‐type viruses, but has little or no inhibitory activity against the reverse transcriptases of feline or murine leukemia viruses. The viruses produced by the SU‐DHL‐1 and SU‐AmB‐3 cell lines have been shown to be infectious for normal human peripheral blood mononuclear cells, normal human bone marrow cells, and certain human lymphoblastoid cell lines. After infection by these viruses, normal human peripheral blood mononuclear cells and human bone marrow cells have exhibited striking changes in growth behavior and morphology which, though not permanently sustained, have many of the features of abortive transformation.

Taxol-oligoarginine conjugates overcome drug resistance in-vitro in human ovarian carcinoma.

OBJECTIVE Multidrug resistance is the major cause of failure of many chemotherapeutic agents. While resistance can arise from several factors, it is often dominated by drug efflux mediated by P-glycoprotein (P-gp), a membrane-bound polysubstrate export pump expressed at high levels in resistant cells. While co-administration of pump inhibitors and a drug could suppress efflux, this two-drug strategy has not yet advanced to therapy. We recently demonstrated that the reversible attachment of a guanidinium-rich molecular transporter, polyarginine, to a drug provides a conjugate that overcomes efflux-based resistance in cells and animals. This study is to determine whether this strategy for overcoming resistance is effective against human disease. METHODS Tumor samples from ovarian cancer patients, both malignant ascites cells and dissociated solid tumor cells, were exposed to Taxol-oligoarginine conjugates designed to release free drug only after cell entry. Cell viability was determined via propidium-iodide uptake by flow cytometry. To analyze bystander effect, toxicity of the drug conjugates was also tested on peripheral blood leucocytes. RESULTS Human ovarian carcinoma specimens resistant to Taxol in vitro demonstrated increased sensitivity to killing by all Taxol-transporter conjugates tested. These studies also show that the drug conjugates were not significantly more toxic to normal human peripheral blood leukocytes than Taxol. CONCLUSIONS These studies with human tumor indicate that oligoarginine conjugates of known drugs can be used to overcome the efflux-based resistance to the drug, providing a strategy that could improve the treatment outcomes of patients with efflux-based drug-resistance.

One step separation of human fetal lymphocytes from nucleated red blood cells

Abstract We describe a one step discontinuous density gradient centrifugation procedure to obtain a 20-fold enrichment of human fetal lymphoid cells, reducing contamination from nucleated red blood cells to less than 1%.

Phase I trial of a novel human monoclonal antibody mAb216 in patients with relapsed or refractory B-cell acute lymphoblastic leukemia.

Background This phase I trial was conducted to determine the safety and pharmacokinetics of monoclonal antibody 216, a human monoclonal Immunoglobulin M antibody targeting a linear B-cell lactosamine antigen, administered alone and in combination with vincristine in patients with relapsed or refractory B-cell acute lymphoblastic leukemia, and to preliminarily assess tumor targeting and efficacy. Design and Methods Three cohorts of patients received escalating doses of monoclonal antibody 216 administered as an intravenous infusion. In the case of poor response to the first dose of monoclonal antibody 216 alone, defined as less than 75% reduction in peripheral blood blast count, a second dose of the antibody with vincristine was given between days 4 and 7. Responses were assessed weekly until day 35. Serum concentration of monoclonal antibody 216 was measured before and after infusion. Monoclonal antibody 216 targeting was determined with an anti-idiotypic antibody to monoclonal antibody 216 and preliminary efficacy was analyzed by changes in peripheral blood blasts. Results Thirteen patients were enrolled. One episode of grade 3 epistaxis was the only dose-limiting toxicity observed. All patients showed a poor response to the first monoclonal antibody 216 infusion with a decrease in peripheral blasts from 6–65% in 9 patients. In 8 patients, addition of vincristine to monoclonal antibody 216 resulted in an average reduction of the peripheral blasts of 81%. One patient without peripheral blasts achieved a hypoplastic marrow without evidence of leukemia after one infusion of monoclonal antibody 216 and monoclonal antibody 216/vincristine each. Monoclonal antibody 216 was detected on peripheral blasts in all patients. Conclusions Treatment with monoclonal antibody 216 in combination with vincristine is feasible and well tolerated in patients with relapsed or refractory B-cell acute lymphoblastic leukemia. Binding of monoclonal antibody 216 to leukemic blasts was efficient, and favorable early responses were observed.