Surgical Stress Abrogates Pre-Existing Protective T Cell Mediated Anti-Tumor Immunity Leading to Postoperative Cancer Recurrence

Anti-tumor CD8⁺ T cells are a key determinant for overall survival in patients following surgical resection for solid malignancies. Using a mouse model of cancer vaccination (adenovirus expressing melanoma tumor-associated antigen (TAA)—dopachrome tautomerase (AdDCT) and resection resulting in major surgical stress (abdominal nephrectomy), we demonstrate that surgical stress results in a reduction in the number of CD8⁺ T cell that produce cytokines (IFNγ, TNFα, Granzyme B) in response to TAA. This effect is secondary to both reduced proliferation and impaired T cell function following antigen binding. In a prophylactic model, surgical stress completely abrogates tumor protection conferred by vaccination in the immediate postoperative period. In a clinically relevant surgical resection model, vaccinated mice undergoing a positive margin resection with surgical stress had decreased survival compared to mice with positive margin resection alone. Preoperative immunotherapy with IFNα significantly extends survival in surgically stressed mice. Importantly, myeloid derived suppressor cell (MDSC) population numbers and functional impairment of TAA-specific CD8⁺ T cell were altered in surgically stressed mice. Our observations suggest that cancer progression may result from surgery-induced suppression of tumor-specific CD8⁺ T cells. Preoperative immunotherapies aimed at targeting the prometastatic effects of cancer surgery will reduce recurrence and improve survival in cancer surgery patients.     

Gene probes to detect cross-culture contamination in hormone producing cell lines

Summary Cross-culture contamination of cell lines propagated in continuous culture is a frequent event and particularly difficult to resolve in cells expressing similar phenotypes. We demonstrate that DNA-DNA hybridization to blotted endonuclease-digested cell DNA effectively detects cross-culture contamination to monitor inter-species as well as intra-species cross contamination. An insulin-producing cell-line, Clone-16, originally cloned from a human fetal endocrine pancreatic cell line did not produce human c-peptide as anticipated. DNA from these cells showed no hybridization to the human ALU sequence probe, BLUR, and lacked restriction fragment length polymorphism typical for the human HLA-DQ β-chain gene. Although a human insulin gene probe showed a weak, nonhuman hybridization pattern, a cDNA probe for the Syrian hamster insulin gene hybridized strongly consistent with a single copy hamster insulin gene. Karyotyping confirmed the absence of human chromosomes in the Clone-16 cells while sizes, centromere indices, and banding patterns were identical to Syrian hamster fibroblasts. We conclude that the insulin-producing Clone-16 cells are of Syrian hamster origin and demonstrate the effective use of gene probes to control the origin of cell cultures. This paper is dedicated to the late Lis Lyngsie in much appreciation of her contributions to this study. This work was supported in part by the National Institutes of Health, Bethesda, MD (grants DK 26190 and 33873). I. Matsuba and B. Michelsen were supported by research fellowships from the Juvenile Diabetes Foundation International, and J. Scholler from the Danish Medical Research Council (J.no. 12-5758).     

Gene product of Moloney murine leukemia virus required for proviral integration is a DNA-binding protein

The 3'-terminal portion of the retroviral pol gene encodes a function required for the formation of the integrated provirus soon after infection of sensitive cells. To permit the isolation of large quantities of the gene product, we expressed various portions of the pol gene of Moloney murine leukemia virus (M-MuLV) as trpE fusion proteins in Escherichia coli. The proteins were found to exhibit strong DNA-binding activity after extraction and renaturation by two different procedures. In the first method, proteins separated by polyacrylamide gel electrophoresis were blotted to nitrocellulose and assayed when bound to the support. The second procedure involved the isolation of proteins in an insoluble fraction, solubilization with guanidine, and renaturation. The characteristics of the binding activity are described and compared with those of authentic viral protein.     

A novel multisite confocal system for rapid Ca2+ imaging from submicron structures in brain slices

In brain slices, resolving fast Ca²⁺ fluorescence signals from submicron structures is typically achieved using 2‐photon or confocal scanning microscopy, an approach that limits the number of scanned points. The novel multiplexing confocal system presented here overcomes this limitation. This system is based on a fast spinning disk, a multimode diode laser and a novel high‐resolution CMOS camera. The spinning disk, running at 20 000 rpm, has custom‐designed spiral pattern that maximises light collection, while rejecting out‐of‐focus fluorescence to resolve signals from small neuronal compartments. Using a 60× objective, the camera permits acquisitions of tens of thousands of pixels at resolutions of ~250 nm per pixel in the kHz range with 14 bits of digital depth. The system can resolve physiological Ca²⁺ transients from submicron structures at 20 to 40 μm below the slice surface, using the low‐affinity Ca²⁺ indicator Oregon Green BAPTA‐5N. In particular, signals at 0.25 to 1.25 kHz were resolved in single trials, or through averages of a few recordings, from dendritic spines and small parent dendrites in cerebellar Purkinje neurons. Thanks to an unprecedented combination of temporal and spatial resolution with relatively simple implementation, it is expected that this system will be widely adopted for multisite monitoring of Ca²⁺ signals.