Vaccination with patient-specific tumor-derived antigen in first remission improves disease-free survival in follicular lymphoma.

PURPOSE: Vaccination with hybridoma-derived autologous tumor immunoglobulin (Ig) idiotype (Id) conjugated to keyhole limpet hemocyanin (KLH) and administered with granulocyte-monocyte colony-stimulating factor (GM-CSF) induces follicular lymphoma (FL) -specific immune responses. To determine the clinical benefit of this vaccine, we conducted a double-blind multicenter controlled phase III trial. PATIENTS AND METHODS: Treatment-naive patients with advanced stage FL achieving complete response (CR) or CR unconfirmed (CRu) after chemotherapy were randomly assigned two to one to receive either Id vaccine (Id-KLH + GM-CSF) or control (KLH + GM-CSF). Primary efficacy end points were disease-free survival (DFS) for all randomly assigned patients and DFS for randomly assigned patients receiving at least one dose of Id vaccine or control. RESULTS: Of 234 patients enrolled, 177 (81%) achieved CR/CRu after chemotherapy and were randomly assigned. For 177 randomly assigned patients, including 60 patients not vaccinated because of relapse (n = 55) or other reasons (n = 5), median DFS between Id-vaccine and control arms was 23.0 versus 20.6 months, respectively (hazard ratio [HR], 0.81; 95% CI, 0.56 to 1.16; P = .256). For 117 patients who received Id vaccine (n = 76) or control (n = 41), median DFS after randomization was 44.2 months for Id-vaccine arm versus 30.6 months for control arm (HR, 0.62; 95% CI, 0.39 to 0.99; P = .047) at median follow-up of 56.6 months (range, 12.6 to 89.3 months). In an unplanned subgroup analysis, median DFS was significantly prolonged for patients receiving IgM-Id (52.9 v 28.7 months; P = .001) but not IgG-Id vaccine (35.1 v 32.4 months; P = .807) compared with isotype-matched control-treated patients. CONCLUSION: Vaccination with patient-specific hybridoma-derived Id vaccine after chemotherapy-induced CR/CRu may prolong DFS in patients with FL. Vaccine isotype may affect clinical outcome and explain differing results between this and other controlled Id-vaccine trials.

CD133+ glioblastoma stem-like cells are radiosensitive with a defective DNA damage response compared with established cell lines.

PURPOSE: CD133+ glioblastoma tumor stem-like cells (TSC) have been defined as radioresistant. However, although previously classified relative to CD133- cells, the radiosensitivity of CD133+ TSCs with respect to the standard glioblastoma model, established glioma cell lines, has not been determined. Therefore, to better understand the radioresponse of this cancer stem cell, we have used established cell lines as a framework for defining their in vitro radioresponse. EXPERIMENTAL DESIGN: The intrinsic radiosensitivity of CD133+ TSC cultures and established glioma cell lines was determined by clonogenic assay. The TSCs and established cell lines were also compared in terms of DNA double-strand break (DSB) repair capacity and cell cycle checkpoint activation. RESULTS: Based on clonogenic analysis, each of the six TSC cultures evaluated was more sensitive to radiation than the established glioma cell lines. Consistent with increased radiosensitivity, the DSB repair capacity as defined by neutral comet assay and gammaH2AX and Rad51 foci was significantly reduced in TSCs compared with the cell lines. Although G2 checkpoint activation was intact, in contrast to the cell lines, DNA synthesis was not inhibited in TSCs after irradiation, indicating the absence of the intra-S-phase checkpoint. CONCLUSIONS: These data indicate that the mechanisms through which CD133+ TSCs respond to radiation are significantly different from those of the traditional glioblastoma in vitro model, established glioma cell lines. If TSCs play a critical role in glioblastoma treatment response, then such differences are likely to be of consequence in the development and testing of radiosensitizing agents.

Physiologic oxygen concentration enhances the stem-like properties of CD133+ human glioblastoma cells in vitro.

In vitro investigations of tumor stem-like cells (TSC) isolated from human glioblastoma (GB) surgical specimens have been done primarily at an atmospheric oxygen level of 20%. To determine whether an oxygen level more consistent with in situ conditions affects their stem cell-like characteristics, we compared GB TSCs grown under conditions of 20% and 7% oxygen. Growing CD133(+) cells sorted from three GB neurosphere cultures at 7% O(2) reduced their doubling time and increased the self-renewal potential as reflected by clonogenicity. Furthermore, at 7% oxygen, the cultures exhibited an enhanced capacity to differentiate along both the glial and neuronal pathways. As compared with 20%, growth at 7% oxygen resulted in an increase in the expression levels of the neural stem cell markers CD133 and nestin as well as the stem cell markers Oct4 and Sox2. In addition, whereas hypoxia inducible factor 1alpha was not affected in CD133(+) TSCs grown at 7% O(2), hypoxia-inducible factor 2alpha was expressed at higher levels as compared with 20% oxygen. Gene expression profiles generated by microarray analysis revealed that reducing oxygen level to 7% resulted in the up-regulation and down-regulation of a significant number of genes, with more than 140 being commonly affected among the three CD133(+) cultures. Furthermore, Gene Ontology categories up-regulated at 7% oxygen included those associated with stem cells or GB TSCs. Thus, the data presented indicate that growth at the more physiologically relevant oxygen level of 7% enhances the stem cell-like phenotype of CD133(+) GB cells.

Bartonella-induced endothelial cell proliferation is mediated by release of calcium from intracellular stores.

The facultative intracellular bacterium Bartonella henselae induces unique angiogenic lesions in immunocompromised hosts. To determine the role of intracellular calcium pools in B. henselae-induced endothelial cell proliferation, we generated B. henselae-conditioned medium (BCM) and tested the ability of these cell-free proteins to induce human umbilical vein endothelial cell (HUVEC) proliferation, CXCL8 production, and intracellular Ca2+ signals. HUVECs incubated with BCM for 3 days had higher cell numbers than controls. In addition, HUVECs produced increased amounts of CXCL8 in response to BCM when compared to medium controls. When BCM was added to HUVECs and the intracellular Ca2+ response measured with the calcium-sensitive dye fura-2/AM, a Ca2+ rise was demonstrated. It was determined that this Ca2+ rise originated from intracellular Ca2+ stores through the use of the Ca2+ ATPase inhibitor thapsigargin. Further, it was demonstrated that BCM enhanced CXCL8 production and HUVEC proliferation in a Ca2+-dependent manner. Conditioned medium from B. henselae causes an intracellular Ca2+ rise in HUVECs, which is involved in B. henselae-induced HUVEC proliferation and CXCL8 production. These results implicate intracellular Ca2+ pools in B. henselae-induced angiogenesis and may lead to increased understanding of the mechanisms of pathogen-induced angiogenesis.

Autocrine role for interleukin-8 in Bartonella henselae-induced angiogenesis.

The gram-negative bacterium Bartonella henselae is capable of causing angiogenic lesions as a result of infection. Previously, it has been shown that B. henselae infection can result in production of the chemokine interleukin-8 (IL-8). In this study, we demonstrated that monocytes, endothelial cells, and hepatocytes produce IL-8 in response to B. henselae infection. We also investigated the role of IL-8 in B. henselae-induced endothelial cell proliferation and capillary tube formation. Both in vitro angiogenesis assays were IL-8 dependent. B. henselae-mediated inhibition of apoptosis, as indicated by gene expression of Bax and Bcl-2, was also shown to be IL-8 dependent in endothelial cells. Furthermore, infection of endothelial cells with B. henselae stimulated upregulation of the IL-8 chemokine receptor CXCR2. Infection of human endothelial cells by B. henselae resulting in IL-8 production likely plays a central role in the ability of this organism to cause angiogenesis during infection.

Interaction of Bartonella henselae with endothelial cells promotes monocyte/macrophage chemoattractant protein 1 gene expression and protein production and triggers monocyte migration.

Bacillary angiomatosis (BA), one of the many clinical manifestations resulting from infection with the facultative intracellular bacterium Bartonella henselae, is characterized by angiogenic lesions. Macrophages have been identified as important effector cells contributing to the angiogenic process during B. henselae infection by infiltrating BA lesions and secreting vascular endothelial growth factor. Monocyte-macrophage chemoattractant protein 1 (MCP-1) recruits macrophages to sites of inflammation. In this study, we investigated the ability of B. henselae to upregulate MCP-1 gene expression and protein production in the human microvascular endothelial cell line HMEC-1. MCP-1 mRNA was induced at 6 and 24 h after treatment with bacteria, whereas protein production was elevated at 6, 24, and 48 h. This induction was not dependent on the presence of bacterial lipopolysaccharide or endothelial cell toll-like receptor 4. However, MCP-1 production was dependent on NF-kappaB activity. Outer membrane proteins of low molecular weight were able to upregulate MCP-1 production. Furthermore, supernatants from B. henselae-infected HMEC-1 were able to induce chemotaxis of THP-1 monocytes. These data suggest a mechanism by which the macrophage effector cell is recruited to the endothelium during B. henselae infection and then contributes to bacterial-induced angiogenesis.