Faculty Bibliography

Human breast carcinoma tumor-infiltrating lymphocytes (TIL) express activation antigens in situ indicative of ongoing immune response-CD28, CD45RO, CD69, CD71, and DR. However, interleukin 2 (IL-2) receptor was poorly expressed: CD25 was detected in only 1/24 samples and CD122 in only 2/24 samples. Furthermore, isolated breast cancer TIL were defective in proliferative response but recover when treated with recombinant IL-2. Nineteen of 24 tumor samples expressed B7-1, B7-2, and CD28 protein, showing that absence of costimulator proteins or counter ligand was not the basis for TIL proliferative deficit. Expression of IL-2 activity was not detected; however, mRNA encoding IL-2 was produced and translatable in vitro. These findings show that human breast cancer tumor-induced repression of IL-2 RNA translation is the basis of failure of TIL to express the IL-2 receptor and subsequent T cell hyporesponsiveness.

An important element in melanoma vaccine construction is to identify peptides from melanoma-associated Ags that have immunogenic potential in humans and are recognized by CD8+ T cells in vivo. To identify such peptides, we evaluated HLA-A*02+ melanoma patients immunized to a polyvalent vaccine containing multiple Ags, including MAGE-3, Melan-A/MART-1, gp100, tyrosinase, melanocortin receptor (MC1R), and dopachrome tautomerase (TRP-2). Using a filter spot assay, we measured peripheral blood CD8+ T cell responses, before and after immunization, to a panel of 45 HLA-A*0201-restricted peptides derived from these Ags. The peptides were selected for immunogenic potential based on their strong binding affinity in vitro to HLA-A*0201. Vaccine treatment induced peptide-specific CD8+ T cell responses to 22 (47.8%) of the peptides. The most striking finding was the HLA-independent heterogeneity of responses to both peptides and Ags. All responding patients reacted to different combination of peptides and Ags even though the responding patients were all A*0201+ and the peptides were all A*0201-restricted. From 9 to 27% of patients developed a CD8+ T cell response to at least one peptide from each Ag, but no more than 3 (14%) reacted to the same peptide from the same Ag. This heterogeneity of responses to individual peptides and Ags in patients with the same haplotype points to the need to construct vaccines of multiple peptides or Ags to maximize the proportion of responding patients

The Escherichia coli NarI restriction enzyme recognition site 5'G1G2C3G4C5C63' is a mutational hotspot for -2 deletions in E. coli plasmid pBR322, resulting in the sequence 5'GGCC3' when G4 is modified by the aromatic amine N-2-(acetyl)aminofluorene (AAF) [Burnouf, D., Koehl, P., and Fuchs, R. P. P. (1995) Proc. Natl. Acad. Sci. U.S.A. 86, 4147-4151] even though each G shows similar reactivity [Fuchs, R. P. P. (1984) J. Mol. Biol. 177, 173-180]. Modification at G4 by the related aromatic amine 2-aminofluorene (AF), which lacks the acetyl group of AAF, can also cause -2 deletions, but at a lower frequency [Bichara, M., and Fuchs, R. P. P. (1985) J. Mol. Biol. 183, 341-351]. A specific mechanism has been proposed to explain the double-base frameshifts in the NarI sequence in which the GC deletion results from a slipped mutagenic intermediate formed during replication [Schaaper, B. M., Koffel-Schwartz, N., and Fuchs, R. P. P. (1990) Carcinogenesis 11, 1087-1095]. We address the following key questions in this study. Why does AAF modification dramatically increase the mutagenicity at the NarI G4 position, and why does AAF enhance the mutagenicity more than AF? We studied two intermediates which model replication at one arm of a fork, using a fragment of DNA modified by AF or AAF at G4 in the NarI sequence: Intermediate I can be converted into intermediate II by misalignment. Elongation of intermediate I leads to error-free translesion synthesis, while elongation of intermediate II leads to a -2 frameshift mutation. Minimized potential energy calculations were carried out using the molecular mechanics program DUPLEX to investigate the conformations of the AF and AAF adducts at G4 in these two intermediates. We find that the slipped mutagenic intermediate is quite stable relative to its normally extended counterpart in the presence of AF and AAF in an abnormal syn orientation of the damaged base. An enhanced probability of elongation from a stable slipped structure rather than a properly aligned one would favor increased -2 frameshift mutations. Furthermore, AAF-modified DNA has a greater tendency to adopt the syn orientation than AF because of its greater bulk, which could explain its greater propensity to cause -2 deletions in the NarI sequence.

BACKGROUND: Many cytokines regulate processes involved in the pathogenesis of proliferative vitreoretinopathy. Transforming growth factor-beta (TGF-beta) is an example of a pluripotent growth factor that regulates cell proliferation, extracellular matrix (ECM) deposition, cell migration, and differentiation--all biological activities involved in the formation and progression of proliferative vitreoretinopathies. METHODS: A review of experimental results that demonstrate how vascular cells generate biologically active TGF-beta is presented. Most cell types--including endothelial cells and pericytes, which form the retinal microvasculature--express TGF-beta as a large latent TGF-beta complex. Mature TGF-beta, the biologically active form, must be generated from the large latent complex before it can signal by binding to its high affinity cell surface receptors. RESULTS: A critical step in regulating TGF-beta effects may be the activation of the large latent TGF-beta complex. Activation of the complex can be achieved by chemical and enzymatic treatments, or by various cell systems. We have identified that co-culturing bovine smooth muscle cells or pericytes and endothelial cells generates active TGF-beta. CONCLUSION: The mechanism of latent TGF-beta activation self-regulates through effectors of plasmin generation. Studying TGF-beta generation by co-cultures of pericytes and endothelial cells can provide us with insights into how disruption of latent TGF-beta activation may lead to unregulated endothelial proliferation, ECM deposition, and cellular infiltration, as observed clinically in neovascular- and fibrotic-related pathologies

Previous attempts at classifying small graft transplants have focused mainly upon graft size and have not taken into consideration other technical factors involved in graft production that may influence the outcome of the surgery. The proposed classification attempts to consider these factors by including various technical aspects of harvesting, dissection, and placement, all of which impact the quality and quantity of the small grafts used in the procedure. By standardizing the nomenclature, as well as the description of the other factors involved in the surgery, communication between physicians and patients may be facilitated. In addition, different procedures may be more accurately studied and compared.

BACKGROUND: The activation of the zymogen plasminogen to the serine protease plasmin by urokinase-type (uPA) and tissue-type (tPA) plasminogen activators (PA) is an important event in a variety of physiologic and pathophysiologic processes in mammals. Enhanced PA activity occurs during angiogenesis and has been correlated in vitro and in vivo with increased tumor aggressiveness and is an indicator of poor prognosis in a variety of tumors in humans. Preliminary studies suggest that the antiulcer drug irsogladine maleate (IM) diminishes PA activity in vitro and may inhibit angiogenesis in vivo. To define the precise mechanism of angiogenesis inhibition by IM in vivo, we tested the ability of IM to blunt angiogenesis in a mouse cornea neovascularization model performed in wild-type and PA-knockout mice. METHODS: Three days prior to pellet implantation, groups of C57Bl/6 wild-type, uPA-deficient (upA-/-), and tPA-deficient (tPA-/-) mice received IM (300 mg/kg), IM (500 mg/kg), or vehicle (0.5% carboxymethylcellulose) via oral gavage. After 3 days of treatment, hydron polymer-coated pellets of sucrose aluminum sulfate containing 100 ng of basic fibroblast growth factor (bFGF) were inserted into surgically created pockets in the cornea of each mouse. On postoperative day 6, the neovascularization of each cornea was evaluated by a blinded observer using slit lamp microscopy and photographed. Angiogenesis was quantified by calculating vascular area (mm2) +/- SEM using a modified formula for a half ellipse that incorporates calibrated vessel measurements [Vessel length (mm) x Clock hours x pi x 0.2]. RESULTS: IM treatment (300 and 500 mg/kg/day) resulted in a dose-dependent reduction of angiogenesis in wild-type mice by 21 and 45.3% (P < 0.02, P < 0.001), in tPA-deficient mice by 42.6 and 46% (P < 0.001, P < 0.001), and in uPA-deficient mice by 27.2 and 46% (P < 0.05, p < 0.001), respectively. No quantitative differences in neovascularization were observed in either treatment group between transgenic mouse strains. No toxicity was noted in any group. CONCLUSION: IM inhibits bFGF-induced angiogenesis in wild-type, tPA-knockout, and uPA-knockout mice. The observation that IM significantly diminishes angiogenesis in both PA-deficient mice and wild-type mice suggests that the mechanism of action of IM may be independent of plasminogen activation.

FGF-2 and VEGF are potent angiogenesis inducers in vivo and in vitro. Here we show that FGF-2 induces VEGF expression in vascular endothelial cells through autocrine and paracrine mechanisms. Addition of recombinant FGF-2 to cultured endothelial cells or upregulation of endogenous FGF-2 results in increased VEGF expression. Neutralizing monoclonal antibody to VEGF inhibits FGF-2-induced endothelial cell proliferation. Endogenous 18-kD FGF-2 production upregulates VEGF expression through extracellular interaction with cell membrane receptors; high-Mr FGF-2 (22-24-kD) acts via intracellular mechanism(s). During angiogenesis induced by FGF-2 in the mouse cornea, the endothelial cells of forming capillaries express VEGF mRNA and protein. Systemic administration of neutralizing VEGF antibody dramatically reduces FGF-2-induced angiogenesis. Because occasional fibroblasts or other cell types present in the corneal stroma show no significant expression of VEGF mRNA, these findings demonstrate that endothelial cell-derived VEGF is an important autocrine mediator of FGF-2-induced angiogenesis. Thus, angiogenesis in vivo can be modulated by a novel mechanism that involves the autocrine action of vascular endothelial cell-derived FGF-2 and VEGF