Complete Genome Sequences of Nine Mycobacteriophages from New Zealand, Beatrix, Carthage, Daegal, Dulcie, Fancypants, Fenn, Inca, Naira, and Robyn.

Beatrix, Carthage, Daegal, Dulcie, Fancypants, Fenn, Inca, Naira, and Robyn are newly isolated bacteriophages capable of infecting Mycolicibacterium smegmatis mc2 155. We discovered, sequenced, and annotated these New Zealand bacteriophages. These phages illustrate that New Zealand harbors a selection of the highly diverse and distributed mycobacteriophage clusters found globally.

Bone marrow stroma-supported culture of T-lineage acute lymphoblastic leukemic cells predicts treatment outcome in children: a Pediatric Oncology Group study.

Significant predictors of treatment outcome are poorly defined for patients with T-lineage acute lymphoblastic leukemia (T-ALL). A high WBC at diagnosis, which has traditionally been a predictor of poor response in T-ALL, has considerably weakened prognostic significance in the face of modern, more intensive chemotherapy. To test the hypothesis that bone marrow stroma-supported leukemic cell recovery might identify children at high risk for relapse, we measured the ex vivo recovery of T-ALL lymphoblasts from 29 newly diagnosed patients using a stromal cell co-culture assay. In all cases the T-ALL lymphoblasts showed an increase in recovery of T-ALL cells (RTC), ranging from 4 to 343%, in comparison to samples maintained without stroma. Since we were blinded to patient outcome in this case-control study, we then correlated patient outcome with RTC. The RTC for 18 patients in complete continuous remission (CCR) for greater than 4 years was stochastically larger than for the 11 patients who eventually relapsed (P = 0.011, by the two-sided Wilcoxon test). Furthermore, 100% of patients with an RTC of more than 26% had a CCR greater than 4 years while 78% of the patients with an RTC of less than 25% relapsed within 4 years. This is the first report to show that higher lymphoblast recovery may predict a more favorable outcome for children with T-ALL. A prospective study is needed to test whether stroma-supported leukemic cell recovery might serve as a basis for assigning risk-adjusted therapy.

Enhanced T-lineage acute lymphoblastic leukaemia cell survival on bone marrow stroma requires involvement of LFA-1 and ICAM-1.

The bone marrow (BM) microenvironment supports leukaemia cell survival and proliferation. The roles played by adhesive receptor interactions in the survival of T-lineage acute lymphoblastic leukaemia (T-ALL) cells on BM stromal cells are not well understood. Recently, we have developed an assay that partially recapitulates the BM microenvironment using HS-5 BM stromal cells. In this assay, the magnitude of ex vivo T-ALL lymphoblast survival predicts patient outcome. We examined the molecular basis for cell-cell adhesive events leading to T-ALL lymphoblast survival on HS-5 and on donor-derived BM stroma. Lympho cyte function-associated antigen-1 (LFA-1) on T-ALL cell lines bound intercellular adhesion molecule-1 (ICAM-1) on HS-5 monolayers, and survival was inhibited 85-98% with monoclonal antibodies directed against LFA-1 or ICAM-1. We compared these results with patient-derived T-ALL lymphoblasts co-cultured on either HS-5 BM or normal BM monolayers and found that LFA-1 and ICAM-1 were required, but not alone sufficient for ex vivo leukaemic cell survival. On normal BM stroma, but not HS-5 monolayers, two additional adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, were highly expressed and contributed to T-ALL cell survival. This is the first report to demonstrate the importance of LFA-1/ICAM-1-mediated adhesion as a critical event in a cascade of cell surface receptor-ligand interactions that regulate T-ALL survival in the BM microenvironment.

Improved quantification of cell survival on stromal monolayers by flow cytometric analyses.

BACKGROUND: The ex vivo survival of leukemic cells maintained on bone marrow stroma is an important tool for the investigation of cell survival and leukemogenesis. Currently, ex vivo survival of leukemic cell survival is measured by coculture on stromal cell monolayers. In these assays, we postulated that two important sources of error might be introduced through either variations in flow volume or in donor stromal cells. METHODS: A previously reported coculture assay that maintains leukemic cells on bone marrow stromal cells was employed. RESULTS: We identified two means of optimizing the coculture assay. First, biologically inert beads having well-characterized fluorescent properties were added to each sample to mathematically adjust for flow-based variations in volume acquisition. The inclusion of fluorescent beads to the basic stromal cell assay showed a significantly lower coefficient of variation as compared to samples analyzed without beads or manually counted using a hemacytometer. Second, in order to minimize variability in bone marrow hematopoietic function between donors, an adherent stromal cell line known to support hematopoiesis (HS-5) was used. When normal human donor stromal cells were used, variability in the survival of leukemic cells was observed on stromal cells derived from different donors. In contrast, statistically significant variability in survival of leukemic cells was not seen on HS-5 monolayers. Finally, we demonstrate that patient-derived leukemic samples may be examined for cell survival using these modifications. CONCLUSIONS: The novel use of fluorescent beads and a hematopoietic-supportive stromal cell line together makes the quantification of stroma-supported cell survival more reproducible, accurate, and amenable to patient-derived samples. These improvements in flow cytometry-based cell quantification are an important step in establishing a role for stromal cell assays in the study of leukemia biology and therapy.