Angiogenic and immune parameters during recombinant interferon-alpha2b adjuvant treatment in patients with melanoma.

As an adjuvant therapy for patients with high risk of recurrent melanoma, high-dose interferon (IFN)-alpha2b therapy has been shown to have some efficacy. We examined 22 patients with resected melanoma who were treated with repeated injections of recombinant IFN-alpha2b during the treatment. Both angiogenic and immune parameters were measured. White blood cells (WBCs) and lymphocyte numbers, lymphocyte subpopulations, serum concentrations of IFN-alpha and anti-IFN-alpha antibodies, and the serum vascular endothelial growth factor (VEGF), interleukin (IL)-8, and basis fibroblast growth factor (bFGF) concentrations were determined over time in resected, recurrence-free patients with American Joint Committee on Cancer (AJCC) stage III melanoma with one or less (LN+ < or = 1, n = 7) or more than one (LN+ > 1, n = 8) lymph nodes involved, and AJCC stage IV resected disease (n = 7). Follow-up and recurrence-free intervals were longer in stage III (LN+ < or = 1) patients compared with stage IV patients (P < 0.05). The number of WBCs and lymphocytes decreased during the treatment for all patient groups (P < 0.001). In addition, percentages of CD8 and CD20 were higher in stage IV patients than in stage III (LN+ > 1) and stage III (LN+ < or = 1) patients at the beginning of therapy (P < 0.05). A significant increase in the percentage of CD20+ cells, mostly B lymphocytes, was observed in the stage III (LN+ > 1) and stage III (LN+ < or = 1) patients over time but not in stage IV patients (P < 0.001). Low IL-8 and bFGF concentrations at the beginning of therapy were associated with significantly longer recurrence-free survival (P < 0.05). These results warrant a larger trial to determine if the differences observed in patients before treatment can provide prognostic markers in patients receiving IFN-alpha2b therapy.

Immune alterations in male and female mice after 2-deoxy-D-glucose administration.

Administration of 2-deoxy-D-glucose (2-DG) induces acute cellular glucoprivation. In the current study, we examined differences in immune parameters after 2-DG administration in both sexes. Male and female BDF1 mice were injected three times, 48 h apart, either with a saline solution (control group) or with 2-DG in saline (500 mg/kg). Two hours after the last injection, blood and spleens were collected. Plasma levels of interleukin-1beta, and interferon-gamma levels were measured. Additionally, the levels of the specific leukocyte antigens CD3, CD4, CD8, T cell receptor (TCR) alpha/beta, I-Ad, and H-2Ld/H-2Db were evaluated by flow cytometry on both blood and spleen cells. The blastogenic response of leukocytes from both tissues to mitogens was assessed. Levels of glucose, corticosterone, testosterone, progesterone, 17beta-estradiol, follicle-stimulating hormone, and luteinizing hormone were also determined. Increases in the percentage of cells bearing TCR alpha/beta and I-Ad in the blood and H-2Ld/H-2Db in the spleen were observed in the 2-DG-treated group for both sexes. In contrast, higher corticosterone and IL-1beta plasma concentrations, as well as higher percentages of splenocytes bearing TCR alpha/beta and I-Ad, and lower mitogen-induced proliferation of mature T splenocytes (79%) were observed in female but not in male mice injected with 2-DG compared with those injected with saline (p < 0.05). Taken together, these results suggest that female mice are more sensitive than male mice to immune alterations induced by 2-DG administration.

Effect of starvation and the viable-but-nonculturable state on green fluorescent protein (GFP) fluorescence in GFP-tagged Pseudomonas fluorescens A506.

The green fluorescent protein (GFP) gene, gfp, of the jellyfish Aequorea victoria is being used as a reporter system for gene expression and as a marker for tracking prokaryotes and eukaryotes. Cells that have been genetically altered with the gfp gene produce a protein that fluoresces when it is excited by UV light. This unique phenotype allows gfp-tagged cells to be specifically monitored by nondestructive means. In this study we determined whether a gfp-tagged strain of Pseudomonas fluorescens continued to fluoresce under conditions under which the cells were starved, viable but nonculturable (VBNC), or dead. Epifluorescent microscopy, flow cytometry, and spectrofluorometry were used to measure fluorescence intensity in starved, VBNC, and dead or dying cells. Results obtained by using flow cytometry indicated that microcosms containing VBNC cells, which were obtained by incubation under stress conditions (starvation at 37.5 degrees C), fluoresced at an intensity that was at least 80% of the intensity of nonstressed cultures. Similarly, microcosms containing starved cells incubated at 5 and 30 degrees C had fluorescence intensities that were 90 to 110% of the intensity of nonstressed cells. VBNC cells remained fluorescent during the entire 6-month incubation period. In addition, cells starved at 5 or 30 degrees C remained fluorescent for at least 11 months. Treatment of the cells with UV light or incubation at 39 or 50 degrees C resulted in a loss of GFP from the cells. There was a strong correlation between cell death and leakage of GFP from the cells, although the extent of leakage varied depending on the treatment. Most dead cells were not GFP fluorescent, but a small proportion of the dead cells retained some GFP at a lower concentration than the concentration in live cells. Our results suggest that gfp-tagged cells remain fluorescent following starvation and entry into the VBNC state but that fluorescence is lost when the cells die, presumably because membrane integrity is lost.