Previously, we showed that the addition of human erythrocyte glycosphingolipids (GSLs) to nonhuman CD4 ؉ or GSL-depleted human CD4؉ cells rendered those cells susceptible to HIV-1 envelope glycoprotein-mediated cell fusion. Individual components in the GSL mixture were isolated by fractionation on a silica-gel column and incorporated into the membranes of CD4 ؉ cells. GSL-supplemented target cells were then examined for their ability to fuse with TF228 cells expressing HIV-1 LAI envelope glycoprotein. We found that one GSL fraction, fraction 3, exhibited the highest recovery of fusion after incorporation into CD4 ؉ nonhuman and GSLdepleted HeLa-CD4 cells and that fraction 3 contained a single GSL fraction. Fraction 3 was characterized by MS, NMR spectroscopy, enzymatic analysis, and immunostaining with an antiglobotriaosylceramide (Gb3) antibody and was found to be Gal(␣134)Gal(134)Glc-Cer (Gb3). The addition of fraction 3 or Gb3 to GSL-depleted HeLa-CD4 cells recovered fusion, but the addition of galactosylceramide, glucosylceramide, the monosialoganglioside, GM3, lactosylceramide, globoside, the disialoganglioside, GD3, or ␣-galactosidase A-digested fraction 3 had no effect. Our findings show that the neutral GSL, Gb3, is required for CD4͞CXCR4-dependent HIV-1 fusion.
Investigators use a surrogate endpoint when the endpoint of interest is too difficult and/or expensive to measure routinely and when they can define some other, more readily measurable, endpoint, that is sufficiently well correlated with the first to justify its use as a substitute. A surrogate endpoint is usually proposed on the basis of a biologic rationale. In cancer studies with survival time as the primary endpoint, surrogate endpoints frequently employed are tumour response, time to progression, or time to reappearance of disease, since these events occur earlier and are unaffected by use of secondary therapies. In early drug development studies, tumour response is often the true primary endpoint. We discuss the investigation of the validity of carcinoembryonic antigen (a tumour marker present in the blood) as a surrogate for tumour response. In considering the validity of surrogate endpoints, one must distinguish between study endpoints that provide a basis for reliable comparisons of therapeutic effect, and clinical endpoints that are useful for patient management but have insufficient sensitivity and/or specificity to provide reproducible assessments of the effects of particular therapies.
Levamisole has been used in a wide array of clinical research and treatment settings over the past two decades, ranging from such diseases as helminthic infestations to various autoimmune diseases. Numerous preclinical evaluations and clinical trials with levamisole in the cancer arena have been sponsored by the National Cancer Institute and other agencies worldwide with the hopes of demonstrating anticancer activity. Trials in advanced breast cancer, lung cancer, colorectal cancer, melanoma, and lymphoproliferative diseases have generally been negative or inconclusive. However, there is some indication that levamisole may be useful by itself as an adjuvant therapy for resected melanoma; recently it has been shown to be effective in combination with fluorouracil (5-FU) as adjuvant therapy for tumor-node-metastasis (TNM) stage III (Dukes' C) colon carcinoma. In the aggregate, the past 20 years of clinical experience with levamisole has resulted in as many questions as answers. However, further testing of the anticancer activity of levamisole can be expected in clinical research trials over the next few years. Hopefully, these future trials will include studies of the mechanisms of action of this agent.
An enzyme-linked immunosorbent spot (ELISPOT) assay for interferon gamma production has been used to analyze specific T cell responses to a Flu 9-mer peptide, and a 9-mer peptide of carcinoembryonic antigen (CEA). Assays were performed on peripheral blood mononuclear cells (PBMC) of HLA-A2-positive patients with CEA-expressing carcinomas, both before and after vaccination with CEA-based vaccines, and from HLA-A2-positive healthy blood donors. The ELISPOT assay utilized aliquots of frozen PBMC, and assays were performed after 24 h in culture with peptide to rule out any artifacts due to long-term in vitro stimulation cycles. An internal standard was used for each assay to define reproducibility of the assay, and all samples from a given patient (pre- and post-vaccination, with both the Flu and CEA peptides) were analyzed simultaneously. The results indicated a trend towards healthy blood donors having higher levels of Flu-specific T cell precursors than do colon carcinoma patients, but these results were not statistically significant (P = 0.06). On the other hand, slightly higher CEA-specific T cell responses were observed in cancer patients with CEA-expressing carcinomas than in healthy blood donors. PBMC from two CEA-based vaccine clinical trials were analyzed for T cell responses to the same CEA peptide and to the Flu control peptide. The first trial consisted of three monthly vaccinations of CEA peptide (designated PPP) in adjuvant. The second trial consisted of cohorts receiving three monthly vaccinations of avipox-CEA recombinant (designated AAA) or cohorts receiving a primary vaccination with recombinant vaccinia-CEA followed by two monthly vaccinations with avipox-CEA (designated VAA). Few, if any, CEA-specific T cell responses were seen in the PPP vaccinations, while the majority of patients receiving the poxvirus CEA recombinants demonstrated increases in CEA-specific T cell responses and no increases in Flu-specific responses. CEA-specific IgG responses were also demonstrated in patients following recombinant CEA poxvirus vaccinations. Statistical analyses of the T cell responses to the same CEA peptide demonstrated a P value of 0.028 for the recombinant poxvirus vaccines, as compared with the peptide vaccine. There were no differences seen (P = 0.37) in Flu-specific responses after these two types of CEA vaccination. These results thus provide the first evidence that poxvirus recombinant-based vaccines are more potent in the initiation of tumor-antigen-specific T cell responses than vaccines employing peptide in adjuvant, when assays are conducted in an identical manner, and in defining responses to the same peptide. These results also demonstrate for the first time that an ELISPOT assay, performed over a 24-h period and without in vitro sensitization, can be successfully used to monitor immune responses to a tumor-associated antigen in cancer patients.
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