Heterogeneous interleukin-15 inducibilities in murine B16 melanoma and RM-1 prostate carcinoma by interferon-alpha treatment.

Long-term treatment of mouse cancer cells with interferon-alpha (IFN-alpha) converts parental B16 melanoma cells to B16alpha vaccine cells. Inoculation of syngeneic mice with UV-irradiated B16alpha vaccine cells triggers immunity to the parental B16 tumor that is mediated by host macrophages, T cells, and NK cells. Lymph node cells from mice inoculated with irradiated B16alpha vaccine cells, but not with irradiated parental cells, proliferate when cultured in vitro, suggesting long-term in vivo activation of lymphoid cells. Both IL-15 mRNA and IL-15 protein are highly induced in B16alpha vaccine cells. The bulk of the induced IL-15 is shown to be cell-associated, either cytoplasmic or membranous. The current study investigated the feasibility of applying the B16alpha vaccination protocol to generate a cancer vaccine against murine RM-1 prostate carcinoma. In comparison to B16alpha vaccine cells, long-term IFN-alpha-treated RM-1 cells (RM-1alpha vaccine cells) showed significant IL-15 mRNA induction but relatively low IL-15 protein up-regulation. When UV-irradiated, a 3-fold increase in intracellular IL-15 was observed in RM-1alpha vaccine cells, suggesting UV damage may have negated a possible control mechanism for IL-15 synthesis. Efficacy of in vivo vaccination of syngeneic mice with UV-irradiated RM-1alpha and B16alpha vaccine cells showed correlation between high IL-15 level and high vaccine efficacy in B16alpha cells compared to low IL-15 level and low vaccine efficacy in RM-1alpha cells. This supports the concept that the induction of IL-15 in tumor cells can be useful for creating whole-cell cancer vaccines.

Hypertrophic versus non hypertrophic scars compared by immunohistochemistry and laser confocal microscopy: type I and III collagens.

Although dermal collagens appear increased in hypertrophic scars, this has not been tested in tissue samples using objective methods. We compared the expression of types I and III collagen in hypertrophic and non hypertrophic scars at 6-12 and 18-24 months after burn using a quantitative method. Among 17 patients with extensive burns, 3 patients had acute scars, 8 had hypertrophic or non hypertrophic scars at 6-12 months after burn and 6 had hypertrophic or non hypertrophic scars at 18-24 months after burn. After clinical assessment of scars using the Vancouver scale, immunohistochemistry for types I and III collagens was performed. Images were captured with a laser scanning confocal microscope and the relative amounts of types I and III collagens were determined in superficial and deep dermis. The effects of time and scar type were assessed using two-way analysis of variance (ANOVA) and Tukey's test. Collagen III scar/normal ratios were higher in hypertrophic scars at both time points (P = 0.05). There were no differences in collagen I scar/normal ratios. Large variation was observed in scars during the acute phase regarding the expression of collagens. Easily accessed by immunohistochemistry and confocal microscopy, type III collagen deposition may help in determining scar phenotype, differentiating hypertrophic and non hypertrophic scars.

Cardiac preconditioning with local laser-induced hyperthermia.

BACKGROUND: Whole body hyperthermia has been shown to be highly effective in preconditioning cardiac tissue against reperfusion injury. The current study was aimed to assess the ability of regional sublethal laser-induced hyperthermia to precondition cardiac tissue against ischemic-induced myocardial infarction. METHODS AND RESULTS: Diode laser radiation was used to locally heat (42 degrees C) the left ventricle by irradiating the epicardial surface of rat heart for 20 min. As control, another group of animals were treated with whole-body hyperthermia (WBH) for 20 min. After a 4-h recovery period, the left coronary artery was occluded for 30 min followed by 4 h of reperfusion. A significant degree of reduction in infarct size as assessed by percent of at-risk area was observed in animals that were subjected to laser-induced local hyperthermia (15.4 +/- 3.1; n = 5) versus the sham group (49.8 +/- 6.6; n = 5). The degree of cardiac protection induced by local hyperthermia was similar to the protection observed when the animals were preconditioned with WBH. Furthermore, regional laser-induced hyperthermia and WBH induced similar degree of up-regulation of heat shock protein 70 in cardiac tissue. Regional hyperthermia up-regulated heat shock protein in regions of the heart beyond that directly subjected to laser-induced heat stress. CONCLUSION: Sublethal local heating of the heart with diode laser irradiation can improve myocardial salvage and may provide a practical approach for tissue preconditioning against reperfusion injury.

IFN-alpha-induced murine B16 melanoma cancer vaccine cells: induction and accumulation of cell-associated IL-15.

Long-term treatment of mouse cancer cells with interferon-alpha (IFN-alpha) converts parental B16 melanoma cells to B16alpha vaccine cells. Inoculation of syngeneic mice with B16alpha vaccine cells triggers immunity to the parental B16 tumor that is mediated by host macrophages, T cells, and natural killer (NK) cells. Lymph node cells from mice inoculated with irradiated B16alpha vaccine cells, but not with irradiated parental cells, proliferate when cultured in vitro, suggesting long-term in vivo activation of lymphoid cells. Long-term IFN-alpha treatment of B16alpha vaccine cells induced both interleukin-15 (IL-15) mRNA and IL-15 protein. The bulk of the induced IL-15 remained cell associated, either cytoplasmic or associated with the cell membrane. Immunofluorescence microscopy studies showed that the cell-associated IL-15 was broadly distributed throughout the cytoplasm. These observations suggest that long-term IFN-alpha treatment may induce primarily the truncated isoform of IL-15. Vaccination with irradiated B16alpha vaccine cells may promote tumor immunity by releasing high levels of cell-associated IL-15 when spontaneously lysed or directly killed by innate immune cells. The release of accumulated cell-associated IL-15 may then trigger a host T cell response to tumor antigens and cause host development of immunity to the B16 tumor cells.

Targeting and penetration of virus receptor bearing cells by nanoparticles coated with envelope proteins of Moloney murine leukemia virus.

One of the most important steps in a productive viral infection is when the virus fuses to a cell membrane and delivers its genome into the cell cytosol. This dynamic event is mediated by interactions between specific virus envelope proteins with their cell-bound receptors. This process is exemplified by Moloney murine leukemia virus (Mo-MLV) where envelope protein interaction with its receptor, mCAT-1, leads to virus-cell membrane fusion and infection of cells. Here, fluorescent nanoparticles (NPs) were coated with Mo-MLV derived membranes (Mo-NPs) by extrusion. Electron microscopy and biochemical analysis showed tight association of the virus membranes and NPs. The coated NPs mimic native virus by binding and entering only cells expressing the virus receptor. Confocal microscopy revealed that the coated NPs were taken up into endocytic compartments containing receptor and were also seen associated with caveolin, a marker of caveolae. To demonstrate that the Mo-NPs could escape endosomes and deliver a protein cargo into the cell cytosol, beta-lactamase (betalac) was covalently coupled to the Mo-NP cores and incubated with cells. betalac activity was only detected in the cytosol of mCAT-1-expressing cells. This is the first time that virus proteins have been used to specifically target NPs to receptor-bearing cells as well as penetration into the cell cytosol. Extrusion provides a rapid, detergent-free method to couple virus membranes to NPs and should be readily applicable for many other virus and NP types.

Whole-body hyperthermia induces up-regulation of vascular endothelial growth factor accompanied by neovascularization in cardiac tissue.

Whole-body hyperthermia (WBH) promotes cardiac protection against ischemia/reperfusion injury, in part by up-regulation of heat shock proteins (HSP). Whether heat stress also promotes up-regulation of angiogenic factors or induces endothelial cell proliferation is unknown. We studied the effects of heat stress on up-regulation of vascular endothelial growth factor (VEGF) and growth of new blood vessels following WBH. Anesthetized rats were subjected to WBH at 42 degrees C for 15 min. The control (n=23) and heated (n=55) groups were allowed to recover for 4, 12, 24, 48, or 72 h prior to harvesting the heart for Western Blot and immunohistochemical assessment of VEGF, HSP70, and platelet endothelial cell adhesion molecular-1 (PECAM-1). A significant increase in VEGF and HSP70 expression was observed as early as 4 h post-heating. The Western Blot analysis revealed a close temporal correlation between up-regulation of HSP70 and VEGF. Maximum VEGF and HSP70 expression occurred at 12 and 24 h post-heating in the left and right ventricles, respectively. The right ventricle showed the greatest expression of both VEGF and HSP70. Immunostaining revealed that VEGF was focally increased in the endothelial cells of capillaries, small arteries, and in interstitium. At 48 and 72 h post-heating, multiple areas of extensive capillary proliferation occurred in the epicardial region of the right ventricle. These observations were verified by quantitative analysis of the density of blood vessels as determined by PECAM-1 staining. Our experiments show that sublethal heat stress can lead to upregulation of both VEGF and HSP70 in cardiac tissue and promote focal endothelial proliferation in the heart.

Identification of membrane-anchoring domains of RLIP76 using deletion mutant analyses.

RLIP76 (RALBP1) is a multifunctional transporter involved in signaling and transmembrane movement of solute allocrites, which include glutathione conjugates and several natural product antineoplastic agents [Awasthi, S., et al. (2000) Biochemistry 39, 9327-9334; (2001) Biochemistry 40, 4159-4168]. Our previous studies suggested that the membrane-anchoring domain resides in the N-terminus of RLIP76, despite the lack of identifiable membrane-spanning domains. Amino acid sequence analysis indicated that this region of RLIP76 contains sequences that are similar to those of vector peptides. We, therefore, have studied the effect of a series of deletion mutant proteins on hydrophobicity and transport activity. RLIP76 or one of its derived deletion mutants was expressed in Escherichia coli, and bacteria were lysed and extracted in buffer without or with the nonionic detergent polidocanol. The ratio of RLIP76 in the detergent/aqueous extracts was found to be 2.5 for the wild-type protein, but decreased to 0.7 in the mutant in which amino acids 154-219 were deleted. Deletion of only one segment of this region (amino acids 171-185) alone resulted in a significant decrease in this ratio to 1.0. For the mutants with deletions within the region from amino acid 154 to 219, loss of hydrophobicity correlated with less incorporation of mutants into artificial liposomes, and decreased transport activity toward doxorubicin and dinitrophenyl-S-glutathione. In contrast, deletion of one of the two ATP-binding sites (at amino acids 65-80 or 415-448) or both sites did not affect hydrophobicity but reduced or abrogated transport activity. NSCLC (H358) stably transfected with del171-185 and del154-219 showed that loss of these regions results in a decrease in the extent of membrane association of RLIP76. Confocal laser immunohistochemistry colocalized amino acids 171-185 with her2/neu on the cell surface. Depletion of wild-type RLIP76 using si-RNA directed to this region in cells transfected with del171-185 resulted in the loss of cell surface expression. These finding demonstrate that amino acids 171-185 constitute a cell surface epitope which is necessary for optimal transport of anthracycline and glutathione conjugates by RLIP76, and that this peptide could be a novel target for antineoplastic therapy.

Interfacial phospholipids inhibit ozone-reactive absorption-mediated cytotoxicity in vitro.

The intrapulmonary distribution of inhaled ozone (O(3)) and induction of site-specific cell injury are related to complex interactions among airflow patterns, local gas-phase concentrations, and the rates of O(3) flux into, and reaction and diffusion within, the epithelial lining fluid (ELF). Recent studies demonstrated that interfacial phospholipid films appreciably inhibited NO(2) absorption. Because surface-active phospholipids are present on alveolar and airway interfaces, we investigated the effects of interfacial films on O(3)-reactive absorption and acute cell injury. Compressed films of dipalmitoyl-glycero-3-phosphocholine (DPPC) and rat lung lavage lipids significantly reduced O(3)-reactive absorption by ascorbic acid, reduced glutathione, and uric acid. Conversely, unsaturated phosphatidylcholine films did not inhibit O(3) absorption. We evaluated O(3)-mediated cell injury using a human lung fibroblast cell culture system, an intermittent tilting exposure regimen to produce a thin covering layer, and nuclear fluorochrome permeability. Exposure produced negligible injury in cells covered with MEM. However, addition of AH(2) produced appreciable (<50%) cell injury. Film spreading of DPPC monolayers necessitated the use of untilted regimens. Induction of acute cell injury in untilted cultures required both AH(2) plus very high O(3) concentrations. Addition of DPPC films significantly reduced cell injury. We conclude that acute cell injury likely results from O(3) reaction with ELF substrates. Furthermore, interfacial films of surface-active, saturated phospholipids reduce the local dose of O(3)-derived reaction products. Finally, because O(3) local dose and tissue damage likely correlate, we propose that interfacial phospholipids may modulate intrapulmonary distribution of inhaled O(3) and the extent of site-specific cell injury.