During angiogenesis, endothelial cells initiate a tissue-invasive program within an interstitial matrix comprised largely of type I collagen. Extracellular matrix–degradative enzymes, including the matrix metalloproteinases (MMPs) MMP-2 and MMP-9, are thought to play key roles in angiogenesis by binding to docking sites on the cell surface after activation by plasmin- and/or membrane-type (MT) 1-MMP–dependent processes. To identify proteinases critical to neovessel formation, an ex vivo model of angiogenesis has been established wherein tissue explants from gene-targeted mice are embedded within a three-dimensional, type I collagen matrix. Unexpectedly, neither MMP-2, MMP-9, their cognate cell-surface receptors (i.e., β3 integrin and CD44), nor plasminogen are essential for collagenolytic activity, endothelial cell invasion, or neovessel formation. Instead, the membrane-anchored MMP, MT1-MMP, confers endothelial cells with the ability to express invasive and tubulogenic activity in a collagen-rich milieu, in vitro or in vivo, where it plays an indispensable role in driving neovessel formation.

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Moloney Murine Leukemia Virus (MoMuLV) causes T cell neoplasms in rodents but is not known to be a pathogen in primates. The core protein and enzyme genes of the MoMuLV genome together with an amphotropic envelope gene are utilized to engineer the cell lines that generate retroviral vectors for use in current human gene therapy applications. We developed a producer clone that generates a very high concentration of retroviral vector particles to optimize conditions for gene insertion into pluripotent hematopoietic stem cells. This producer cell line also generates a much lower concentration of replication-competent virus that arose through recombination. Stem cells from rhesus monkeys were purified by immunoselection with an anti-CD34 antibody, incubated in vitro for 80-86 h in the presence of retroviral vector particles with accompanying replication-competent virus and used to reconstitute recipients whose bone marrow had been ablated by total body irradiation. The retroviral vector genome was detected in circulating cells of five of eight transplant recipients of CD34+ cells and in the circulating cells of two recipients of infected, unfractionated bone marrow mononuclear cells. Three recipients of CD34+ cells had a productive infection with replication-competent virus. Six or seven mo after transplantation, each of these animals developed a rapidly progressive T cell neoplasm involving the thymus, lymph nodes, liver, spleen, and bone marrow. Lymphoma cells contained 10-50 copies of the replication-competent virus, but lacked the retroviral vector genome. We conclude that replication-competent viruses arising from producer cells making retroviral vectors can be pathogenic in primates, which underscores the importance of carefully screening retroviral producer clones used in human trials to exclude contamination with replication-competent virus.

More effective therapeutic strategies are required for patients with poor-prognosis systemic sclerosis (SSc). A phase 2 single-arm study of high-dose immunosuppressive therapy (HDIT) and autologous CD34-selected hematopoietic cell transplantation (HCT) was conducted in 34 patients with diffuse cutaneous SSc. HDIT included total body irradiation (800 cGy) with lung shielding, cyclophosphamide (120 mg/kg), and equine antithymocyte globulin (90 mg/kg). Neutrophil and platelet counts were recovered by 9 (range, 7 to 13) and 11 (range, 7 to 25) days after HCT, respectively. Seventeen of 27 (63%) evaluable patients who survived at least 1 year after HDIT had sustained responses at a median follow-up of 4 (range, 1 to 8) years. There was a major improvement in skin (modified Rodnan skin score, ؊22.08; P < .001) and overall function (modified Health Assessment Questionnaire Disability Index, ؊1.03; P < .001) at final evaluation. Importantly, for the first time, biopsies confirmed a statistically significant decrease of dermal fibrosis compared with baseline (P < .001). Lung, heart, and kidney func-